Measurement of Intracellular Ions by Flow Cytometry
UNIT 9.8
Avery D. Posey, Jr.,1 Omkar U. Kawalekar,1 and Carl H. June1 1
Abramson Family Cancer Research Institute, and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania
Using flow cytometry, single-cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. Most earlier techniques for measuring cellular activation parameters determined the mean value for a population of cells, which did not permit optimal resolution of the responses. The flow cytometer is particularly useful for this purpose because it can measure ion concentrations in large numbers of single cells and thereby allows ion concentration to be correlated with other parameters such as immunophenotype and cell cycle stage. A limitation of flow cytometry, however, is that it does not permit resolution of certain complex kinetic responses such as cellular oscillatory responses. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the C 2015 by John Wiley & Sons, principles of data analysis and interpretation. Inc. Keywords: calcium analysis r ion analysis r flow cytometry
How to cite this article: Posey, A.D. Jr., Kawalekar, O.U., and June, C.H. 2015. Measurement of intracellular ions by flow cytometry. Curr. Protoc. Cytom. 72:9.8.1-9.8.21. doi: 10.1002/0471142956.cy0908s72
INTRODUCTION The flow cytometer can be used to measure various functional parameters that are of increasing interest to immunologists. The development of a number of fluorescent probes permits the measurement of various intracellular free ion concentrations in single living cells. Among these ions are calcium, magnesium, sodium, potassium, and hydrogen (pH). Most previously available techniques to measure cellular activation parameters determined the mean value for a population of cells, but this did not permit optimal resolution of the responses. The flow cytometer is a particularly useful instrument for this purpose because it permits the measurement of ion concentrations in large numbers of single cells and allows correlation with other parameters such as immunophenotype and cell cycle (Oh-hora et al., 2013). In many cases, there is marked heterogeneity in the changes that occur, even in cells that were previously thought to be homogeneous. The flow cytometer does not, however, permit the resolution of some complex kinetic responses such as cellular oscillatory responses (Osipchuk and Cahalan, 1992; Allbritton and Meyer, 1993; Allen et al., 2001). For this purpose, video microscopy with digital image analysis is required, a technique that is complementary to flow cytometry for the study of various parameters of cell activation (Monteith, 2000; Dewitt et al., 2003). In this unit, a flow cytometric protocol using the UV-excitable calcium indicator Indo-1 is described (Basic Protocol). Alternative protocols describe methods to measure high cellular calcium concentrations (Alternate Protocol 1), use of visual light excitable
Current Protocols in Cytometry 9.8.1-9.8.21, April 2015 Published online April 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142956.cy0908s72 C 2015 John Wiley & Sons, Inc. Copyright
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calcium indicators Fluo-3, Fluo-4, Rhod-2, eFluor514, and Fura Red (Alternate Protocol 2), and the use of a spectrophotometer to measure intraceullar calcium concentrations in the absence of a flow cytometer (Alternate Protocol 3). Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay (Support Protocol 1), and methods to facilitate dye loading (Support Protocol 2). BASIC PROTOCOL
USE OF INDO-1 AND FLOW CYTOMETRY TO MEASURE CELLULAR CALCIUM CONCENTRATION In this protocol, intracellular ionized calcium concentration ([Ca2+ ]i ) is measured using Indo-1 dye and ratiometric analysis. All commercially available flow cytometers can be used to perform this assay, provided the instrument is capable of UV illumination. In addition, on many instruments, cells may be electronically sorted based on a particular calcium response and cultured for later analysis. The protocol can be divided into three stages: preparation of cells to be analyzed, set up of the flow cytometer, and data analysis and display. The protocol requires expertise in basic flow cytometric techniques.
Materials Murine splenic lymphocytes or human peripheral blood lymphocytes Cell loading medium (HBSS or similar, 1 mM Ca2+ , 1 mM Mg2+ , 1% FBS) 2 mg/ml Indo-1 acetoxymethyl ester (Indo-1 AM; see recipe) 100 mM probenecid (see recipe) 1 mg/ml ionomycin (see recipe) Dimethyl sulfoxide (DMSO; Sigma) or 10% bleach in water Saline or phosphate-buffered saline (PBS) Beckman TJ-6 rotor (or equivalent) 12 × 75–mm polypropylene tubes (BD Biosciences, cat. no. 2063) 30° or 37°C water bath Fluorescence microscope Flow cytometer with UV light source and heated sample chamber (e.g., Becton Dickinson LSRFortessa), and software for kinetic and ratiometric analysis (e.g., FlowJo) Load cells with Indo-1 1. Collect lymphocytes in polypropylene tubes and centrifuge 5 min in a Beckman TJ-6 rotor at 1200 rpm (300 × g), room temperature. Resuspend the pellet in cell loading medium at 106 to 107 cells/ml. Use murine splenic lymphocytes or human peripheral blood lymphocytes in initial experiments, as they are easily and reliably loaded. Later, when the other aspects of the technique are validated on the flow cytometer, both adherent and non-adherent cells can be analyzed. Use polypropylene tubes to minimize loss of cells.
2. Add 2 mg/ml Indo-1 AM to 2 μg/ml final. Incubate 30 min at 30° or 37°C. The cells are loaded with the membrane-permeant form (acetoxymethyl ester) of Indo-1 (Indo-1 AM). Cellular esterases cause the trapping of this highly charged moiety in the cells. Typically, 20% of the Indo-1 becomes trapped and concentrated within the cell (Chused et al., 1987).
Flow Cytometric Calcium Analysis
The optimal conditions for loading must be empirically determined for each cell type. Rates of loading of the Indo-1 AM vary between cell types, especially as a consequence of variations in intracellular esterase activity. More rapid loading rates are seen in platelets and monocytes than in lymphocytes, and in growing cell lines rather than resting cells. More uniform loading is often observed if pluronic F-127 is included together with Indo-1 AM (see Support Protocol 2). However, this must be validated for each cell type as the
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use of pluronic F-127 actually inhibits flux in some systems (e.g. thymocytes). In addition, incubation at 30°C can aid loading of cells which tend to compartmentalize the Indo-1. An optional step is to stain an aliquot of Indo-1-loaded cells for simultaneous immunofluorescence analysis. This is done with saturating amounts of azide-free, non-UV excited fluorescent antibodies (e.g. FITC, PE, Alexa 488) (as determined by titration experiments or by manufacturer’s recommendations). Incubate 20 min at room temperature. This step may be done at 4°C to minimize antigen modulation, but after chilling, cells may require an extended equilibration time at 37°C to return to physiologic functioning for the calcium assay.
3. Optional: Add 100 mM probenecid to 4 mM final during loading. Probenecid may improve cellular loading by minimizing leakage of Indo-1 and cell-tocell variation in dye content (Vandenberghe and Ceuppens, 1990; Baus et al., 1994). A protocol for preparation of stock solutions of probenecid was first described in Di Virgilio et al. (1990) and updated reagents are now available (see Reagents and Solutions). Life Technologies recommends the use of an optimized formulation of nonionic surfactants like plurionic-127 (PowerLoad Concentrate; cat. no. P10020) in combination with probenecid for maximal dye loading.
4. Centrifuge the cells for 5 min at 1200 rpm (300 × g), room temperature. Gently resuspend (DO NOT VORTEX) cells in cell loading medium at the desired cell concentration (3 × 106 cells/ml). Store cells at room temperature and protect from light until analysis. It is often preferable to let cells rest for 15 min before starting analysis. This presumably allows complete conversion of the calcium-insensitive form of Indo-1 ester into the calcium-sensitive (charged) form of Indo-1 and enhances cell uptake of additional equilibrating calcium to compensate for Indo-1-bound calcium.
Set up the flow cytometer 5. Set up and adjust flow cytometer. Use a violet bandpass filter centered at 395 nm ± 10 nm and a blue bandpass filter centered at 510 nm ± 20 nm, along with a 505 nm beamsplitter to separate the violet from the blue signal to improve the ratio (see Critical Parameters). 6. Set light scatter gates and optimize photomultiplier tube gain settings by placing the mean blue fluorescence in the upper half of the histogram channels and the violet fluorescence in the lower half of the histogram channels. Use linear rather than logarithmic amplification, and gate out dead cells with light scatter and violet fluorescence windows (see Critical Parameters). 7. Check instrument setup and cellular loading by treating 1 × 105 cells in cell loading medium with 1 mg/ml ionomycin to 1 to 2 μg/ml final. An immediate response in 100% of cells should occur. Since ionomycin may adhere to the tubing, carefully wash ionomycin from the system. Flush the lines for 1 min with DMSO or 10% bleach for 2 min followed by deionized water for 2 min, followed by a 1 min wash with cell loading medium to bind any residual ionomycin. It is critical that each experiment include a determination of R, the ratio of violet/blue fluorescence of resting cells and Rmax , the ratio of violet/blue fluorescence of cells after stimulation by the calcium ionophore ionomycin. If the instrument is properly aligned and the cells loaded adequately, the ratio of Rmax :R is 6 to 9 (see Troubleshooting). Use fluorescence microscopy to verify quality of Indo-1 loading; compartmentation of Indo-1 is indicated by the presence of punctate dots of fluorescence.
8. To calibrate Indo-1 fluorescence ratio to [Ca2+ ]i , suspend cells in a series of calciumEGTA buffers and ionomycin (Chused et al., 1987). See Support Protocol 1 for details.
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Analyze Indo-1-loaded cells 9. Warm an aliquot of Indo-1-loaded cells for 5 to 10 min at 37°C before analysis. Use 5 × 105 cells per 10-min assay. 10. Analyze the cells at 37°C in cell loading medium. Use saline or PBS for the sheath fluid. The rate of cell analysis can vary. Commonly, cells are analyzed at 200 to 300 cells/sec. A higher flow rate may be required when sorting cells or when analyzing a rare event, such as a calcium signal in a small subset of cells identified by antibody staining (see Critical Parameters).
11. Optional: Set regions for simultaneous immunofluorescence analysis. Combining the use of multiple fluorescent probes with Indo-1 analysis allows determination of [Ca2+ ]i in complex immunophenotypic subsets. Limiting the analysis of Indo-1 fluorescence to uniquely defined immunophenotypic gates allows information relating to each identifiable cellular subset to be derived from a single sample.
12. Optional: If desired, sort cells on the basis of [Ca2+ ]i responses. Sorting on the basis of Indo-1 fluorescence can be an important tool for the selection and identification of genetic variants in the biochemical pathways leading to Ca2+ mobilization and cell growth and differentiation (see background information). ALTERNATE PROTOCOL 1
FLOW CYTOMETRIC APPROACHES TO MEASURE HIGH CELLULAR CALCIUM CONCENTRATIONS Materials 2 mg/ml Indo-5 F acetoxymethyl ester (Indo-5 F AM; AnaSpec, cat. no. 84051) 2 mg/ml Mag-Indo-1 acetoxymethyl ester (Mag-Indo-1 AM; Life Technologies, cat. no. M-1295) The Kd of Indo-1 is 230 nm, and thus it is saturated at calcium concentrations above 1μM. Probes have been developed with diminished calcium-binding affinity that can be used in settings of high calcium concentration (Gee et al., 2000a). Indo-5 F is an analog of Indo-1 that has a Kd of 470 nm. Except for the change in the Ca2+ concentration response range, the Ca2+ -dependent spectral shifts produced by Indo-5 F are essentially identical to those of Indo-1, and the probe uses the same optical filter sets. Approaches for cellular loading, analysis and calibration of responses are the same as for Indo-1. Mag-Indo-1 AM, which has a Kd of 35μM for Ca2+ and 2.7 mM for Mg2+ can be used for measuring intracellular Ca2+ levels between 1 μM and 100 μM (Chien et al., 1999; Pesco et al., 2001).
ALTERNATE PROTOCOL 2
Flow Cytometric Calcium Analysis
SIMULTANEOUS USE OF VISUAL LIGHT EXCITABLE CALCIUM INDICATORS AND FURA RED FLUORESCENCE RATIOS FOR FLOW CYTOMETRIC CALCIUM MEASUREMENT Several calcium-sensitive probes are also available that can be excited with light in the visible range, so that all flow cytometers can, in principle, measure calcium. Fluo-3 is a fluorescein-based calcium probe developed by Tsien and colleagues (Minta et al., 1989) and it has a Kd of 390 nm. Fluo-4, with its minor structural modification (two chlorines substituted by fluorines), binds to calcium with similar affinity but has a substantially higher fluorescence output than Fluo-3, both dyes that emit in the green emission group (Gee et al., 2000b). Similar to Fluo-3 and Fluo-4, eFluor514 Calcium Sensor Dye emits in the green light range but has a higher calcium affinity with a Kd of 232 nm. In the orange emission group, Rhod-2 has a Kd of 570 nm, although previously reported as higher (Minta et al., 1989), and a fluorescence output similar to Fluo-3 (Takahashi et al., 1999). Rhod-2 exhibits a mitochondrial subcellular location (Rutter et al., 1996). The use
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of these probes allows the flow cytometric measurement of calcium on instruments that are not equipped with a UV light source. However, because Fluo-3, Fluo-4, and Rhod-2 are nonratiometric, they are difficult to calibrate. In addition, the binding of proteins, and a number of intracellular variables can affect the Kd and fluorescence output of some of these indicators (Harkins et al., 1993). However, for ratiometric measurements, these indicators can also be co-loaded into cells with other Ca2+ -sensitive probes. We favor the combined use of the near-infrared Fura Red, which exhibits reciprocal shifts in fluorescence intensity upon calcium binding. With this protocol, a high-sensitivity, low noise assay for calcium changes can be done using dyes that are excited at 488 nm (Lipp and Niggli, 1993; Novak and Rabinovitch, 1994). Previously, the Indo-1 based ratiometric assay using UV illumination was required for flow cytometric assays to detect small subsets of responding cells. The combined cellular loading with two nonratiometric calcium probes, Fura Red and Fluo-3, Fluo-4, eFluor514, or Rhod-2 permits a sensitive ratiometric assay using visual illumination. Lower-affinity calcium indicators based on Fluo-3, Fluo-4, and Rhod-2 are also available to investigate intracellular calcium concentrations in the micromolar range.
Additional Materials (also see the Basic Protocol) 10 mg/ml Fluo-3 (Life Technologies, cat. no. F1241) or Fluo-4 (Life Technologies, cat. no. F23917) or eFluor514 Calcium Sensor Dye (Affymetrix, cat. no. 65-0859) or Rhod-2 (Life Technologies, cat. no. R1245 MP) 10 mg/ml Fura Red acetoxymethyl ester (Fura Red AM; see recipe) Appropriately labeled antibody Load cells with Fluo-3 and Fura Red 1. Collect the cells in polypropylene tube and centrifuge for 5 min at 1200 rpm (300 × g), room temperature. Resuspend the pellet in cell loading medium at 106 to 107 cells/ml. 2. Add 10 mg/ml Fluo-3 AM to 4 μg/ml final and Fura Red AM to 10 μg/ml final. Incubate 30 min at 30° to 37°C. The amounts of Fluo-3 and Fura Red may require adjustment; the goal to achieve simultaneous loading with balanced emissions from the two probes. Since Fura Red has relatively weak fluorescence, about 2.5 to 3 times more Fura Red that Fluo-3 should be used (Lipp and Niggli, 1993; Schild et al., 1994). Inclusion of pluronic F-127 may improve loading (must be determined empirically; see Support Protocol 2).
3. Optional: Add 100 mM probenecid to 4 mM final during loading to delay leakage of probes. A protocol for preparation of stock solutions of probenecid is described in Reagents and Solutions and usage described in Support Protocol 2.
4. Centrifuge the cells for 5 min at 300 × g, room temperature. Gently resuspend the cells in cell loading medium at the desired cell concentration (3 × 106 /ml). Store cells at room temperature and protect from light until analysis. It is recommended that analysis be performed as soon after loading as possible. Leakage and compartmentalization of the probes is accelerated if cells are stored at 37°C.
5. Optional: Stain cells with appropriately labeled antibody, if desired. There is spectral overlap between the Fura Red and PE fluorescence emissions; compensation is required to separate the signals. In a typical 4-color benchtop analyzer, a 635 nm red diode laser is also present and can be used to excite allophycocyanin (APC) as an alternative surface label for use in this protocol. More advanced cytometers offer more options for co-staining.
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Set up flow cytometer 6. Excite fluorescence with the 488-nm line of an argon ion laser. Collect Fluo-3 fluorescence at 515 to 535 nm and Fura Red emission at 665 to 685 nm using linear amplification. The instrument must be equipped with a means to analyze cells that are maintained at 37°C. Analyze Fluo-3 and Fura Red-loaded cells 7. Analyze cells by gating on forward and right angle light scatter. Exclude dead cells by gating out cells without Fluo-3 fluorescence. Collect Fluo-3 fluorescence versus time, and Fura Red fluorescence versus time. Use software to analyze the Fluo-3/Fura Red ratio versus time, similar to Indo-1 data analysis (see Basic Protocol).
8. Determine adequacy of cellular loading and instrumental alignment by treating a sample of cells with 1 mg/ml ionomycin to 2 μg/ml final as in step 7 of the Basic Protocol. If 8. Calculate Ca2+ from the equation given in the commentary. When determining Rmin , it is necessary to add EGTA to Triton-permeabilized cells and to raise the pH to >8; the affinity of EGTA for calcium is highly pH-dependent, and in acid medium, a true Rmin may not be obtained. In preliminary tests, determine the amount of Tris base required to titrate the pH of the medium to pH 8.3 Alternatively, use BAPTA, a selective calcium chelator that displays pH-independent calcium binding (Life Technologies, cat. no. B-1212). Carefully wash Triton and ionomycin from cuvettes between replicate experiments. It is necessary to measure autofluorescence of unloaded cells (in presence and absence of Triton) and to subtract “background” fluorescence from that attributable to Indo-1 or Fluo-3.
USE OF CALCIUM:EGTA BUFFERS TO CALIBRATE FLOW CYTOMETRIC CALCIUM MEASUREMENTS
SUPPORT PROTOCOL 1
This protocol employs a series of precisely prepared calcium buffer solutions to perform an in situ calibration in cells. Indo-1 loaded cells are suspended in the calcium buffers containing metabolic inhibitors in order to overcome cellular calcium homeostasis. The protocol (Chused et al., 1987; Li et al., 1987) is carried out with the aid of commercially prepared calcium buffer kits. It is not possible to use methods to directly prepare a solution that contains calcium in a concentration similar to that found inside living cells due to the contamination of laboratory water by calcium (generally micromolar amounts). In
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addition, it is not possible to prepare solutions with sufficient precision using gravimetric methods because CaCl2 and EGTA each contain variable amounts of water (Miller and Smith, 1984). Therefore, buffers of known free calcium concentration are useful for experiments in which cells are maintained at a particular calcium concentration and for standardization of Indo-1 fluorescence ratio changes. The approach is to prepare solutions of known calcium concentration. This is done by mixing binary buffer solutions consisting of buffer A (an equimolar solution of calcium and a calcium chelator such as EGTA) and buffer B, (identical to buffer A except that it lacks calcium) in a set of metabolic poisons. EGTA or BAPTA are used as the chelator because they have high selectivity for calcium over magnesium, an ion that is 104 times more abundant than calcium in the cytoplasm, and therefore can be used to control calcium in the presence of physiologic concentrations of magnesium. To obtain a buffer with a desired calcium concentration, the experimental conditions (temperature, pH, magnesium concentration, and ionic strength) are entered into a set of equations, and buffers A and B are mixed at the necessary ratio.
Materials Calcium Calibration Buffer Kit #1, zero, and 10 mM CaEGTA (10 mM K2 EGTA and 10 mM CaEGTA, Life Technologies, cat. no. C-3008MP) Phosphate-buffered saline (PBS) with 20 mM HEPES, pH 7.20, without calcium or magnesium containing the following cellular poisons (use care, as these are highly toxic reagents): Ionomycin 1 mg/ml stock solution in dimethyl sulfoxide (DMSO) to 3 μg/ml final Nigericin 10 mg/ml stock solution in methanol to 2.0 μg/ml final Carbonyl cyanide m-chlorophenylhydrazone (CCCP) 1 mM stock solution in dimethyl sulfoxide (DMSO) at 10 μM final 2-deoxyglucose 1 M stock solution in water, 40 mM final Sodium azide 3 M stock solution in water, 60 mM final 37°C incubator Additional reagents and equipment for loading mouse or human lymphocytes with Indo−1, or Fluo−3 plus Fura Red in PBS containing poisons (see Chused et al., 1987) Preparation of Calcium:EGTA buffers 1. Life Technologies supplies a kit to make buffers of 11 different Ca2+ concentrations for the evaluation of [Ca2+ ] from the fluorescence intensity measurements of the calcium indication of choice in specific experimental environmental conditions. Calcium indicator dye is added to a zero calcium buffer (10 mM K2 EGTA) and a high Ca2+ buffer (10 mM CaEGTA). Using reciprocal dilutions, create a series of 11 buffers with 1 mM steps in [Ca2+ ]. Follow the protocol provided by Life Technologies (http://tools.lifetechnologies.com/content/sfs/manuals/CalciumCalibrationBufferKits _PI.pdf) for specific details on the kit. Do not use glassware in the preparation of the calcium buffers, as this may be a source of calcium contamination. Be sure to use the salts of these dyes (Life Technologies, cat. nos. I-1202 and F-3715, respectively) and not the esters; the esters will not bind Ca2+ without intracellular esterases, absent in this protocol.
Flow Cytometric Calcium Analysis
It is important to note that factors affect free calcium concentration in complex aqueous solutions, see Pethig et al. (1989); Oiki et al. (1994); McGuigan et al. (2007); Bers et al. (2010); and Poenie (1990). The complexity of these calculations makes use of a computer highly desirable. Maxchelator, a computer program that will determine ionized calcium and magnesium concentrations as a function of pH, total
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calcium and magnesium concentrations, temperature, and ionic strength is available on the at http://maxchelator.stanford.edu.
2. If desired, check buffers by measuring the actual free calcium concentration with Indo-1 or Fluo-3 as described in Alternate Protocol 3. Use flow cytometer for in situ calibration assay.
3. Follow the protocol of Chused and coworkers (1987). Load mouse or human lymphocytes with Indo-1, or Fluo-3 plus Fura Red in PBS with 20 mM HEPES, pH 7.20, containing ionomycin, nigericin, CCCP, 2-deoxyglucose, and sodium azide. Modify loading to omit serum.
4. Wash the cells once and resuspend aliquots of 5 × 105 cells in 1 ml each of buffers 1 to 11. Take care to assure that there is no serum carryover; serum will buffer calcium, and any factor that affects pH, temperature, viscosity and ionic strength of the buffer will change the calcium concentration of the buffers. The cells are “clamped” in the presence of metabolic inhibitors and ionophores, so that the calcium concentration of the buffer should be the same as the cell interior.
5. Incubate cells at 37°C for 90 min to permit equilibration of cells. Equilibrium should be achieved within 1 to 2 hr. See Chused et al. (1987), Negulescu and Machen (1990), and Thomas and Delaville (1991) for details.
6. Analyze the cells to determine the steady-state fluorescence ratio (for Indo-1 or Fluo3 plus Fura Red-loaded cells) of cells in each buffer solution. Some dead cells should be apparent, reflecting the effects of the cellular poisons. Plot the peak ratio channel (for Indo-1 or Fluo-3 plus Fura Red-loaded cells) versus calcium of each buffer. See Figure 9.8.1 for example. 1200
Indo-1 ratio (channel number)
1000
800
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400
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0 1
10
100
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10,000
Ca2+ concentration (nM)
Figure 9.8.1 Example of in situ calibration of Indo-1 ratio shifts. Human T cells were loaded with Indo-1 and electrochemical gradients disabled as described in the text. The cells were suspended in a series of calcium buffers ranging from 6 nM to 22 μM and steady-state Indo-1 fluorescence ratios determined. The ratio of the 22-μM sample was off-scale, and was > channel 1024.
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The free calcium concentration of each buffer is dependent on the pH. If the pH is not 7.20, the numbers will differ from those given in the Life Technologies protocol, and the calcium concentration must be calculated as described above. SUPPORT PROTOCOL 2
USE OF PLURONIC DETERGENT F-127 TO LOAD CELLS WITH INDO-1 OR FLUO-3 OR FLUO-3 AND FURA RED When using cell lines or cells other than lymphocytes, it is not uncommon to have difficulty with proper loading, as evidenced by poor shifts after cellular stimulation. The basis for poor loading is commonly due to compartmentalization and incomplete hydrolysis of the dye ester. The use of pluronic detergent may sometimes circumvent this difficulty (Poenie et al., 1986; Lanza et al., 1987; Vandenberghe and Ceuppens, 1990), and with some cell types, causes a spectacular improvement in signaling. In addition, there is less heterogeneity in cell-to-cell Indo-1 and Fluo-3 uptake after loading in the presence of pluronic F-127. Again, pluronic does not work for all cell types and should be tested before use.
Additional Materials (also see the Basic Protocol) 20% (w/v) pluronic F-127 in DMSO (Life Technologies, cat. no. P-3000 MP; alternatively, use PowerLoad Concentrate, cat. no. P10020) 100 mM probenecid (Life Technologies, cat. no. P36400), optional Fetal bovine serum (FBS; heat-inactivated 1 hr, 56°C) 1. Suspend cells at 5 × 106 cells/ml in 1 ml cell loading medium (HBSS containing 1% FBS, 1 mM Ca2+ , 1 mM Mg2+ ). 2. To a 50 μg vial of Indo-1, add 25 μl of 20% pluronic F-127, and 113 μl FBS. Mix and allow 5 min for Indo-1 to dissolve. Prepare a similar mixture if using Fluo-3 or Fura Red. 3. Optional: Add 100 mM probenecid to 4 mM final during loading. Probenecid may improve cellular loading by minimizing leakage of organic ionic dyes and cell-to-cell variation in dye content (Vandenberghe and Ceuppens, 1990; Baus et al., 1994). Sulfinpyrazone will also work.
4. Add 5 to 8 μl of the Indo-1 mixture from step 2 to the cells. Incubate 30 min at 30°C. The final concentration of Indo-1 and pluronic F-127 is 1.8 μM and 0.02% respectively. Slightly more or less Indo-1 mixture may be required, depending on the batch of Indo-1 and on the cell type.
5. Wash the cells by centrifuging 5 min at 300 × g, room temperature. Resuspend in medium of choice at 3 × 106 cells/ml for analysis beginning with step 5 in the Basic Protocol.
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Cell loading medium Hanks balanced salt solution (HBSS) containing 1 mM calcium, 1 mM magnesium, 1% FBS, heat-inactivated 1 hr at 56°C or 0.5% bovine serum albumin (BSA). Store up to 1 month at 4°C. Fluo-3 AM (similar for Fluo-4, eFluor514, and Rhod-2), 10 mg/ml Flow Cytometric Calcium Analysis
Dissolve Fluo-3 (Life Technologies, cat. no. F1241) at 10 mg/ml in dimethyl sulfoxide (DMSO) and store in a desiccator in the dark up to 1 week at −20°C.
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The molecular weight of Fluo-3 ester is 1130 g/mol. The concentrations of each indicator necessary must be determined empirically, focusing on matching the brightness of indicator 1 and Fura Red.
Fura Red AM, 10 mg/ml Dissolve Fura Red acetoxymethyl ester (Fura Red AM; Life Technologies, cat. no. F-3020) at 10 mg/ml in dimethyl sulfoxide (DMSO) and store in a desiccator in the dark up to 1 week at −20°C. The molecular weight of Fura Red ester is 945 g/mol.
Indo-1 AM (similar for Indo-5 F AM and Mag-Indo-1 AM), 2 mg/ml Dissolve Indo-1 acetoxymethyl ester (Indo-1 AM; Life Technologies cat. no. I-1223) at 2 mg/ml in dimethyl sulfoxide (DMSO) and store in a desiccator in the dark up to several months at −20°C. For a 50 μg aliquot of Indo-1 AM, add 25 μl DMSO. The molecular weight of Indo-1 ester is 1009.9 g/mol; a 2 mg/ml stock is almost exactly 2 mM.
Ionomycin, 1 mg/ml Dissolve at 1 mg/ml ionomycin (Millipore, cat. no. 407950) in dimethyl sulfoxide (DMSO) or 100% (v/v) ethanol. Use of DMSO minimizes evaporation of solvent and consequent concentration of ionomycin. Store up to 1 year at 4°C. Probenecid, 100 mM Life Technologies’ water-soluble probenecid (cat. no. P36400) removes the necessity for 1 M NaOH to dissolve it. The contents of one vial of 77 mg water-soluble probenecid and can be dissolved in 2.5 ml HBSS buffer for 100 mM final stock concentration. Otherwise, probenecid is relatively insoluble in aqueous solution, unless alkalinized and must be dissolved in water, adding 1 M NaOH until dissolved. The pH should be 9 to 10. COMMENTARY Background Information In their resting state, eukaryotic cells maintain an internal calcium ion concentration that is far below that of the extracellular environment. Ionized calcium has an important role as a mediator of transmembrane signal transduction, and elevations in intracellular ionized calcium concentration ([Ca2+ ]i ) regulate diverse cellular processes (Berridge, 2012). Thus, measurement of [Ca2+ ]i in living cells is of considerable interest to investigators in immunology and cell biology. Until 1980, measurement of [Ca2+ ]i was restricted to large invertebrate cells where the use of microelectrodes was possible. Subsequently, bioluminescent indicators such as aequorin, a calcium-activated photoprotein, were described, but were limited in their application by the necessity of loading cells by microinjection or other forms of membrane disruption (Blinks et al., 1982; Cobbold and Rink, 1987). Tsien and colleagues (Tsien et al., 1982) developed Quin-2, making it possible for the first
time to measure [Ca2+ ]i in virtually any population of cells. Unfortunately, Quin-2 has a relatively low extinction coefficient and quantum yield, and this, in conjunction with the fact that it does not have useful ratiometric properties, made the detection of calcium responses in single cells impractical. A second family of dyes was developed with the invention of Fura-2 and Indo-1 (Grynkiewicz et al., 1985), and it became possible to measure [Ca2+ ]i in single cells of almost any type. Fura-2 is best suited for applications involving fluorescence microscopy with digital image analysis, while Indo-1, due to its unique fluorescence properties, is best suited for flow cytometry. With Indo-1, there is a 6fold increase in signal when cells change from basal levels of [Ca2+ ]i to saturating amounts of [Ca2+ ]i . In addition, the ratio of Indo-1 signals allows measurement of [Ca2+ ]i that is independent of cell size or brightness. Cells loaded with Indo-1 exhibit a 3-fold range in brightness, and after measuring the ratio of
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Indo-1 calcium-bound to calcium-unbound at the proper wavelengths, the cell-to-cell variation in the signal from resting cells has a coefficient of variation of only 5% to 10% (Rabinovitch et al., 1986). Thus, with Indo-1 it is possible to discriminate responses of small subpopulations of cells within a larger population of non-responding cells. Flow cytometric cell conjugate assays have also been developed so that the calcium signals that occur during antigen presentation or target cell recognition can be studied using Indo-1 (Abe et al., 1992; Alexander et al., 1992; Van Graft et al., 1993). Another advantage of Indo-1 is sensitivity of response. Results of artificial mixing experiments with Jurkat T leukemia and K562 myeloid leukemia cell lines indicated that subpopulations of cells with variant [Ca2+ ]i comprising
UNIT 9.8
Avery D. Posey, Jr.,1 Omkar U. Kawalekar,1 and Carl H. June1 1
Abramson Family Cancer Research Institute, and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania
Using flow cytometry, single-cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. Most earlier techniques for measuring cellular activation parameters determined the mean value for a population of cells, which did not permit optimal resolution of the responses. The flow cytometer is particularly useful for this purpose because it can measure ion concentrations in large numbers of single cells and thereby allows ion concentration to be correlated with other parameters such as immunophenotype and cell cycle stage. A limitation of flow cytometry, however, is that it does not permit resolution of certain complex kinetic responses such as cellular oscillatory responses. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the C 2015 by John Wiley & Sons, principles of data analysis and interpretation. Inc. Keywords: calcium analysis r ion analysis r flow cytometry
How to cite this article: Posey, A.D. Jr., Kawalekar, O.U., and June, C.H. 2015. Measurement of intracellular ions by flow cytometry. Curr. Protoc. Cytom. 72:9.8.1-9.8.21. doi: 10.1002/0471142956.cy0908s72
INTRODUCTION The flow cytometer can be used to measure various functional parameters that are of increasing interest to immunologists. The development of a number of fluorescent probes permits the measurement of various intracellular free ion concentrations in single living cells. Among these ions are calcium, magnesium, sodium, potassium, and hydrogen (pH). Most previously available techniques to measure cellular activation parameters determined the mean value for a population of cells, but this did not permit optimal resolution of the responses. The flow cytometer is a particularly useful instrument for this purpose because it permits the measurement of ion concentrations in large numbers of single cells and allows correlation with other parameters such as immunophenotype and cell cycle (Oh-hora et al., 2013). In many cases, there is marked heterogeneity in the changes that occur, even in cells that were previously thought to be homogeneous. The flow cytometer does not, however, permit the resolution of some complex kinetic responses such as cellular oscillatory responses (Osipchuk and Cahalan, 1992; Allbritton and Meyer, 1993; Allen et al., 2001). For this purpose, video microscopy with digital image analysis is required, a technique that is complementary to flow cytometry for the study of various parameters of cell activation (Monteith, 2000; Dewitt et al., 2003). In this unit, a flow cytometric protocol using the UV-excitable calcium indicator Indo-1 is described (Basic Protocol). Alternative protocols describe methods to measure high cellular calcium concentrations (Alternate Protocol 1), use of visual light excitable
Current Protocols in Cytometry 9.8.1-9.8.21, April 2015 Published online April 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142956.cy0908s72 C 2015 John Wiley & Sons, Inc. Copyright
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calcium indicators Fluo-3, Fluo-4, Rhod-2, eFluor514, and Fura Red (Alternate Protocol 2), and the use of a spectrophotometer to measure intraceullar calcium concentrations in the absence of a flow cytometer (Alternate Protocol 3). Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay (Support Protocol 1), and methods to facilitate dye loading (Support Protocol 2). BASIC PROTOCOL
USE OF INDO-1 AND FLOW CYTOMETRY TO MEASURE CELLULAR CALCIUM CONCENTRATION In this protocol, intracellular ionized calcium concentration ([Ca2+ ]i ) is measured using Indo-1 dye and ratiometric analysis. All commercially available flow cytometers can be used to perform this assay, provided the instrument is capable of UV illumination. In addition, on many instruments, cells may be electronically sorted based on a particular calcium response and cultured for later analysis. The protocol can be divided into three stages: preparation of cells to be analyzed, set up of the flow cytometer, and data analysis and display. The protocol requires expertise in basic flow cytometric techniques.
Materials Murine splenic lymphocytes or human peripheral blood lymphocytes Cell loading medium (HBSS or similar, 1 mM Ca2+ , 1 mM Mg2+ , 1% FBS) 2 mg/ml Indo-1 acetoxymethyl ester (Indo-1 AM; see recipe) 100 mM probenecid (see recipe) 1 mg/ml ionomycin (see recipe) Dimethyl sulfoxide (DMSO; Sigma) or 10% bleach in water Saline or phosphate-buffered saline (PBS) Beckman TJ-6 rotor (or equivalent) 12 × 75–mm polypropylene tubes (BD Biosciences, cat. no. 2063) 30° or 37°C water bath Fluorescence microscope Flow cytometer with UV light source and heated sample chamber (e.g., Becton Dickinson LSRFortessa), and software for kinetic and ratiometric analysis (e.g., FlowJo) Load cells with Indo-1 1. Collect lymphocytes in polypropylene tubes and centrifuge 5 min in a Beckman TJ-6 rotor at 1200 rpm (300 × g), room temperature. Resuspend the pellet in cell loading medium at 106 to 107 cells/ml. Use murine splenic lymphocytes or human peripheral blood lymphocytes in initial experiments, as they are easily and reliably loaded. Later, when the other aspects of the technique are validated on the flow cytometer, both adherent and non-adherent cells can be analyzed. Use polypropylene tubes to minimize loss of cells.
2. Add 2 mg/ml Indo-1 AM to 2 μg/ml final. Incubate 30 min at 30° or 37°C. The cells are loaded with the membrane-permeant form (acetoxymethyl ester) of Indo-1 (Indo-1 AM). Cellular esterases cause the trapping of this highly charged moiety in the cells. Typically, 20% of the Indo-1 becomes trapped and concentrated within the cell (Chused et al., 1987).
Flow Cytometric Calcium Analysis
The optimal conditions for loading must be empirically determined for each cell type. Rates of loading of the Indo-1 AM vary between cell types, especially as a consequence of variations in intracellular esterase activity. More rapid loading rates are seen in platelets and monocytes than in lymphocytes, and in growing cell lines rather than resting cells. More uniform loading is often observed if pluronic F-127 is included together with Indo-1 AM (see Support Protocol 2). However, this must be validated for each cell type as the
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use of pluronic F-127 actually inhibits flux in some systems (e.g. thymocytes). In addition, incubation at 30°C can aid loading of cells which tend to compartmentalize the Indo-1. An optional step is to stain an aliquot of Indo-1-loaded cells for simultaneous immunofluorescence analysis. This is done with saturating amounts of azide-free, non-UV excited fluorescent antibodies (e.g. FITC, PE, Alexa 488) (as determined by titration experiments or by manufacturer’s recommendations). Incubate 20 min at room temperature. This step may be done at 4°C to minimize antigen modulation, but after chilling, cells may require an extended equilibration time at 37°C to return to physiologic functioning for the calcium assay.
3. Optional: Add 100 mM probenecid to 4 mM final during loading. Probenecid may improve cellular loading by minimizing leakage of Indo-1 and cell-tocell variation in dye content (Vandenberghe and Ceuppens, 1990; Baus et al., 1994). A protocol for preparation of stock solutions of probenecid was first described in Di Virgilio et al. (1990) and updated reagents are now available (see Reagents and Solutions). Life Technologies recommends the use of an optimized formulation of nonionic surfactants like plurionic-127 (PowerLoad Concentrate; cat. no. P10020) in combination with probenecid for maximal dye loading.
4. Centrifuge the cells for 5 min at 1200 rpm (300 × g), room temperature. Gently resuspend (DO NOT VORTEX) cells in cell loading medium at the desired cell concentration (3 × 106 cells/ml). Store cells at room temperature and protect from light until analysis. It is often preferable to let cells rest for 15 min before starting analysis. This presumably allows complete conversion of the calcium-insensitive form of Indo-1 ester into the calcium-sensitive (charged) form of Indo-1 and enhances cell uptake of additional equilibrating calcium to compensate for Indo-1-bound calcium.
Set up the flow cytometer 5. Set up and adjust flow cytometer. Use a violet bandpass filter centered at 395 nm ± 10 nm and a blue bandpass filter centered at 510 nm ± 20 nm, along with a 505 nm beamsplitter to separate the violet from the blue signal to improve the ratio (see Critical Parameters). 6. Set light scatter gates and optimize photomultiplier tube gain settings by placing the mean blue fluorescence in the upper half of the histogram channels and the violet fluorescence in the lower half of the histogram channels. Use linear rather than logarithmic amplification, and gate out dead cells with light scatter and violet fluorescence windows (see Critical Parameters). 7. Check instrument setup and cellular loading by treating 1 × 105 cells in cell loading medium with 1 mg/ml ionomycin to 1 to 2 μg/ml final. An immediate response in 100% of cells should occur. Since ionomycin may adhere to the tubing, carefully wash ionomycin from the system. Flush the lines for 1 min with DMSO or 10% bleach for 2 min followed by deionized water for 2 min, followed by a 1 min wash with cell loading medium to bind any residual ionomycin. It is critical that each experiment include a determination of R, the ratio of violet/blue fluorescence of resting cells and Rmax , the ratio of violet/blue fluorescence of cells after stimulation by the calcium ionophore ionomycin. If the instrument is properly aligned and the cells loaded adequately, the ratio of Rmax :R is 6 to 9 (see Troubleshooting). Use fluorescence microscopy to verify quality of Indo-1 loading; compartmentation of Indo-1 is indicated by the presence of punctate dots of fluorescence.
8. To calibrate Indo-1 fluorescence ratio to [Ca2+ ]i , suspend cells in a series of calciumEGTA buffers and ionomycin (Chused et al., 1987). See Support Protocol 1 for details.
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Analyze Indo-1-loaded cells 9. Warm an aliquot of Indo-1-loaded cells for 5 to 10 min at 37°C before analysis. Use 5 × 105 cells per 10-min assay. 10. Analyze the cells at 37°C in cell loading medium. Use saline or PBS for the sheath fluid. The rate of cell analysis can vary. Commonly, cells are analyzed at 200 to 300 cells/sec. A higher flow rate may be required when sorting cells or when analyzing a rare event, such as a calcium signal in a small subset of cells identified by antibody staining (see Critical Parameters).
11. Optional: Set regions for simultaneous immunofluorescence analysis. Combining the use of multiple fluorescent probes with Indo-1 analysis allows determination of [Ca2+ ]i in complex immunophenotypic subsets. Limiting the analysis of Indo-1 fluorescence to uniquely defined immunophenotypic gates allows information relating to each identifiable cellular subset to be derived from a single sample.
12. Optional: If desired, sort cells on the basis of [Ca2+ ]i responses. Sorting on the basis of Indo-1 fluorescence can be an important tool for the selection and identification of genetic variants in the biochemical pathways leading to Ca2+ mobilization and cell growth and differentiation (see background information). ALTERNATE PROTOCOL 1
FLOW CYTOMETRIC APPROACHES TO MEASURE HIGH CELLULAR CALCIUM CONCENTRATIONS Materials 2 mg/ml Indo-5 F acetoxymethyl ester (Indo-5 F AM; AnaSpec, cat. no. 84051) 2 mg/ml Mag-Indo-1 acetoxymethyl ester (Mag-Indo-1 AM; Life Technologies, cat. no. M-1295) The Kd of Indo-1 is 230 nm, and thus it is saturated at calcium concentrations above 1μM. Probes have been developed with diminished calcium-binding affinity that can be used in settings of high calcium concentration (Gee et al., 2000a). Indo-5 F is an analog of Indo-1 that has a Kd of 470 nm. Except for the change in the Ca2+ concentration response range, the Ca2+ -dependent spectral shifts produced by Indo-5 F are essentially identical to those of Indo-1, and the probe uses the same optical filter sets. Approaches for cellular loading, analysis and calibration of responses are the same as for Indo-1. Mag-Indo-1 AM, which has a Kd of 35μM for Ca2+ and 2.7 mM for Mg2+ can be used for measuring intracellular Ca2+ levels between 1 μM and 100 μM (Chien et al., 1999; Pesco et al., 2001).
ALTERNATE PROTOCOL 2
Flow Cytometric Calcium Analysis
SIMULTANEOUS USE OF VISUAL LIGHT EXCITABLE CALCIUM INDICATORS AND FURA RED FLUORESCENCE RATIOS FOR FLOW CYTOMETRIC CALCIUM MEASUREMENT Several calcium-sensitive probes are also available that can be excited with light in the visible range, so that all flow cytometers can, in principle, measure calcium. Fluo-3 is a fluorescein-based calcium probe developed by Tsien and colleagues (Minta et al., 1989) and it has a Kd of 390 nm. Fluo-4, with its minor structural modification (two chlorines substituted by fluorines), binds to calcium with similar affinity but has a substantially higher fluorescence output than Fluo-3, both dyes that emit in the green emission group (Gee et al., 2000b). Similar to Fluo-3 and Fluo-4, eFluor514 Calcium Sensor Dye emits in the green light range but has a higher calcium affinity with a Kd of 232 nm. In the orange emission group, Rhod-2 has a Kd of 570 nm, although previously reported as higher (Minta et al., 1989), and a fluorescence output similar to Fluo-3 (Takahashi et al., 1999). Rhod-2 exhibits a mitochondrial subcellular location (Rutter et al., 1996). The use
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of these probes allows the flow cytometric measurement of calcium on instruments that are not equipped with a UV light source. However, because Fluo-3, Fluo-4, and Rhod-2 are nonratiometric, they are difficult to calibrate. In addition, the binding of proteins, and a number of intracellular variables can affect the Kd and fluorescence output of some of these indicators (Harkins et al., 1993). However, for ratiometric measurements, these indicators can also be co-loaded into cells with other Ca2+ -sensitive probes. We favor the combined use of the near-infrared Fura Red, which exhibits reciprocal shifts in fluorescence intensity upon calcium binding. With this protocol, a high-sensitivity, low noise assay for calcium changes can be done using dyes that are excited at 488 nm (Lipp and Niggli, 1993; Novak and Rabinovitch, 1994). Previously, the Indo-1 based ratiometric assay using UV illumination was required for flow cytometric assays to detect small subsets of responding cells. The combined cellular loading with two nonratiometric calcium probes, Fura Red and Fluo-3, Fluo-4, eFluor514, or Rhod-2 permits a sensitive ratiometric assay using visual illumination. Lower-affinity calcium indicators based on Fluo-3, Fluo-4, and Rhod-2 are also available to investigate intracellular calcium concentrations in the micromolar range.
Additional Materials (also see the Basic Protocol) 10 mg/ml Fluo-3 (Life Technologies, cat. no. F1241) or Fluo-4 (Life Technologies, cat. no. F23917) or eFluor514 Calcium Sensor Dye (Affymetrix, cat. no. 65-0859) or Rhod-2 (Life Technologies, cat. no. R1245 MP) 10 mg/ml Fura Red acetoxymethyl ester (Fura Red AM; see recipe) Appropriately labeled antibody Load cells with Fluo-3 and Fura Red 1. Collect the cells in polypropylene tube and centrifuge for 5 min at 1200 rpm (300 × g), room temperature. Resuspend the pellet in cell loading medium at 106 to 107 cells/ml. 2. Add 10 mg/ml Fluo-3 AM to 4 μg/ml final and Fura Red AM to 10 μg/ml final. Incubate 30 min at 30° to 37°C. The amounts of Fluo-3 and Fura Red may require adjustment; the goal to achieve simultaneous loading with balanced emissions from the two probes. Since Fura Red has relatively weak fluorescence, about 2.5 to 3 times more Fura Red that Fluo-3 should be used (Lipp and Niggli, 1993; Schild et al., 1994). Inclusion of pluronic F-127 may improve loading (must be determined empirically; see Support Protocol 2).
3. Optional: Add 100 mM probenecid to 4 mM final during loading to delay leakage of probes. A protocol for preparation of stock solutions of probenecid is described in Reagents and Solutions and usage described in Support Protocol 2.
4. Centrifuge the cells for 5 min at 300 × g, room temperature. Gently resuspend the cells in cell loading medium at the desired cell concentration (3 × 106 /ml). Store cells at room temperature and protect from light until analysis. It is recommended that analysis be performed as soon after loading as possible. Leakage and compartmentalization of the probes is accelerated if cells are stored at 37°C.
5. Optional: Stain cells with appropriately labeled antibody, if desired. There is spectral overlap between the Fura Red and PE fluorescence emissions; compensation is required to separate the signals. In a typical 4-color benchtop analyzer, a 635 nm red diode laser is also present and can be used to excite allophycocyanin (APC) as an alternative surface label for use in this protocol. More advanced cytometers offer more options for co-staining.
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Set up flow cytometer 6. Excite fluorescence with the 488-nm line of an argon ion laser. Collect Fluo-3 fluorescence at 515 to 535 nm and Fura Red emission at 665 to 685 nm using linear amplification. The instrument must be equipped with a means to analyze cells that are maintained at 37°C. Analyze Fluo-3 and Fura Red-loaded cells 7. Analyze cells by gating on forward and right angle light scatter. Exclude dead cells by gating out cells without Fluo-3 fluorescence. Collect Fluo-3 fluorescence versus time, and Fura Red fluorescence versus time. Use software to analyze the Fluo-3/Fura Red ratio versus time, similar to Indo-1 data analysis (see Basic Protocol).
8. Determine adequacy of cellular loading and instrumental alignment by treating a sample of cells with 1 mg/ml ionomycin to 2 μg/ml final as in step 7 of the Basic Protocol. If 8. Calculate Ca2+ from the equation given in the commentary. When determining Rmin , it is necessary to add EGTA to Triton-permeabilized cells and to raise the pH to >8; the affinity of EGTA for calcium is highly pH-dependent, and in acid medium, a true Rmin may not be obtained. In preliminary tests, determine the amount of Tris base required to titrate the pH of the medium to pH 8.3 Alternatively, use BAPTA, a selective calcium chelator that displays pH-independent calcium binding (Life Technologies, cat. no. B-1212). Carefully wash Triton and ionomycin from cuvettes between replicate experiments. It is necessary to measure autofluorescence of unloaded cells (in presence and absence of Triton) and to subtract “background” fluorescence from that attributable to Indo-1 or Fluo-3.
USE OF CALCIUM:EGTA BUFFERS TO CALIBRATE FLOW CYTOMETRIC CALCIUM MEASUREMENTS
SUPPORT PROTOCOL 1
This protocol employs a series of precisely prepared calcium buffer solutions to perform an in situ calibration in cells. Indo-1 loaded cells are suspended in the calcium buffers containing metabolic inhibitors in order to overcome cellular calcium homeostasis. The protocol (Chused et al., 1987; Li et al., 1987) is carried out with the aid of commercially prepared calcium buffer kits. It is not possible to use methods to directly prepare a solution that contains calcium in a concentration similar to that found inside living cells due to the contamination of laboratory water by calcium (generally micromolar amounts). In
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addition, it is not possible to prepare solutions with sufficient precision using gravimetric methods because CaCl2 and EGTA each contain variable amounts of water (Miller and Smith, 1984). Therefore, buffers of known free calcium concentration are useful for experiments in which cells are maintained at a particular calcium concentration and for standardization of Indo-1 fluorescence ratio changes. The approach is to prepare solutions of known calcium concentration. This is done by mixing binary buffer solutions consisting of buffer A (an equimolar solution of calcium and a calcium chelator such as EGTA) and buffer B, (identical to buffer A except that it lacks calcium) in a set of metabolic poisons. EGTA or BAPTA are used as the chelator because they have high selectivity for calcium over magnesium, an ion that is 104 times more abundant than calcium in the cytoplasm, and therefore can be used to control calcium in the presence of physiologic concentrations of magnesium. To obtain a buffer with a desired calcium concentration, the experimental conditions (temperature, pH, magnesium concentration, and ionic strength) are entered into a set of equations, and buffers A and B are mixed at the necessary ratio.
Materials Calcium Calibration Buffer Kit #1, zero, and 10 mM CaEGTA (10 mM K2 EGTA and 10 mM CaEGTA, Life Technologies, cat. no. C-3008MP) Phosphate-buffered saline (PBS) with 20 mM HEPES, pH 7.20, without calcium or magnesium containing the following cellular poisons (use care, as these are highly toxic reagents): Ionomycin 1 mg/ml stock solution in dimethyl sulfoxide (DMSO) to 3 μg/ml final Nigericin 10 mg/ml stock solution in methanol to 2.0 μg/ml final Carbonyl cyanide m-chlorophenylhydrazone (CCCP) 1 mM stock solution in dimethyl sulfoxide (DMSO) at 10 μM final 2-deoxyglucose 1 M stock solution in water, 40 mM final Sodium azide 3 M stock solution in water, 60 mM final 37°C incubator Additional reagents and equipment for loading mouse or human lymphocytes with Indo−1, or Fluo−3 plus Fura Red in PBS containing poisons (see Chused et al., 1987) Preparation of Calcium:EGTA buffers 1. Life Technologies supplies a kit to make buffers of 11 different Ca2+ concentrations for the evaluation of [Ca2+ ] from the fluorescence intensity measurements of the calcium indication of choice in specific experimental environmental conditions. Calcium indicator dye is added to a zero calcium buffer (10 mM K2 EGTA) and a high Ca2+ buffer (10 mM CaEGTA). Using reciprocal dilutions, create a series of 11 buffers with 1 mM steps in [Ca2+ ]. Follow the protocol provided by Life Technologies (http://tools.lifetechnologies.com/content/sfs/manuals/CalciumCalibrationBufferKits _PI.pdf) for specific details on the kit. Do not use glassware in the preparation of the calcium buffers, as this may be a source of calcium contamination. Be sure to use the salts of these dyes (Life Technologies, cat. nos. I-1202 and F-3715, respectively) and not the esters; the esters will not bind Ca2+ without intracellular esterases, absent in this protocol.
Flow Cytometric Calcium Analysis
It is important to note that factors affect free calcium concentration in complex aqueous solutions, see Pethig et al. (1989); Oiki et al. (1994); McGuigan et al. (2007); Bers et al. (2010); and Poenie (1990). The complexity of these calculations makes use of a computer highly desirable. Maxchelator, a computer program that will determine ionized calcium and magnesium concentrations as a function of pH, total
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calcium and magnesium concentrations, temperature, and ionic strength is available on the at http://maxchelator.stanford.edu.
2. If desired, check buffers by measuring the actual free calcium concentration with Indo-1 or Fluo-3 as described in Alternate Protocol 3. Use flow cytometer for in situ calibration assay.
3. Follow the protocol of Chused and coworkers (1987). Load mouse or human lymphocytes with Indo-1, or Fluo-3 plus Fura Red in PBS with 20 mM HEPES, pH 7.20, containing ionomycin, nigericin, CCCP, 2-deoxyglucose, and sodium azide. Modify loading to omit serum.
4. Wash the cells once and resuspend aliquots of 5 × 105 cells in 1 ml each of buffers 1 to 11. Take care to assure that there is no serum carryover; serum will buffer calcium, and any factor that affects pH, temperature, viscosity and ionic strength of the buffer will change the calcium concentration of the buffers. The cells are “clamped” in the presence of metabolic inhibitors and ionophores, so that the calcium concentration of the buffer should be the same as the cell interior.
5. Incubate cells at 37°C for 90 min to permit equilibration of cells. Equilibrium should be achieved within 1 to 2 hr. See Chused et al. (1987), Negulescu and Machen (1990), and Thomas and Delaville (1991) for details.
6. Analyze the cells to determine the steady-state fluorescence ratio (for Indo-1 or Fluo3 plus Fura Red-loaded cells) of cells in each buffer solution. Some dead cells should be apparent, reflecting the effects of the cellular poisons. Plot the peak ratio channel (for Indo-1 or Fluo-3 plus Fura Red-loaded cells) versus calcium of each buffer. See Figure 9.8.1 for example. 1200
Indo-1 ratio (channel number)
1000
800
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0 1
10
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10,000
Ca2+ concentration (nM)
Figure 9.8.1 Example of in situ calibration of Indo-1 ratio shifts. Human T cells were loaded with Indo-1 and electrochemical gradients disabled as described in the text. The cells were suspended in a series of calcium buffers ranging from 6 nM to 22 μM and steady-state Indo-1 fluorescence ratios determined. The ratio of the 22-μM sample was off-scale, and was > channel 1024.
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The free calcium concentration of each buffer is dependent on the pH. If the pH is not 7.20, the numbers will differ from those given in the Life Technologies protocol, and the calcium concentration must be calculated as described above. SUPPORT PROTOCOL 2
USE OF PLURONIC DETERGENT F-127 TO LOAD CELLS WITH INDO-1 OR FLUO-3 OR FLUO-3 AND FURA RED When using cell lines or cells other than lymphocytes, it is not uncommon to have difficulty with proper loading, as evidenced by poor shifts after cellular stimulation. The basis for poor loading is commonly due to compartmentalization and incomplete hydrolysis of the dye ester. The use of pluronic detergent may sometimes circumvent this difficulty (Poenie et al., 1986; Lanza et al., 1987; Vandenberghe and Ceuppens, 1990), and with some cell types, causes a spectacular improvement in signaling. In addition, there is less heterogeneity in cell-to-cell Indo-1 and Fluo-3 uptake after loading in the presence of pluronic F-127. Again, pluronic does not work for all cell types and should be tested before use.
Additional Materials (also see the Basic Protocol) 20% (w/v) pluronic F-127 in DMSO (Life Technologies, cat. no. P-3000 MP; alternatively, use PowerLoad Concentrate, cat. no. P10020) 100 mM probenecid (Life Technologies, cat. no. P36400), optional Fetal bovine serum (FBS; heat-inactivated 1 hr, 56°C) 1. Suspend cells at 5 × 106 cells/ml in 1 ml cell loading medium (HBSS containing 1% FBS, 1 mM Ca2+ , 1 mM Mg2+ ). 2. To a 50 μg vial of Indo-1, add 25 μl of 20% pluronic F-127, and 113 μl FBS. Mix and allow 5 min for Indo-1 to dissolve. Prepare a similar mixture if using Fluo-3 or Fura Red. 3. Optional: Add 100 mM probenecid to 4 mM final during loading. Probenecid may improve cellular loading by minimizing leakage of organic ionic dyes and cell-to-cell variation in dye content (Vandenberghe and Ceuppens, 1990; Baus et al., 1994). Sulfinpyrazone will also work.
4. Add 5 to 8 μl of the Indo-1 mixture from step 2 to the cells. Incubate 30 min at 30°C. The final concentration of Indo-1 and pluronic F-127 is 1.8 μM and 0.02% respectively. Slightly more or less Indo-1 mixture may be required, depending on the batch of Indo-1 and on the cell type.
5. Wash the cells by centrifuging 5 min at 300 × g, room temperature. Resuspend in medium of choice at 3 × 106 cells/ml for analysis beginning with step 5 in the Basic Protocol.
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Cell loading medium Hanks balanced salt solution (HBSS) containing 1 mM calcium, 1 mM magnesium, 1% FBS, heat-inactivated 1 hr at 56°C or 0.5% bovine serum albumin (BSA). Store up to 1 month at 4°C. Fluo-3 AM (similar for Fluo-4, eFluor514, and Rhod-2), 10 mg/ml Flow Cytometric Calcium Analysis
Dissolve Fluo-3 (Life Technologies, cat. no. F1241) at 10 mg/ml in dimethyl sulfoxide (DMSO) and store in a desiccator in the dark up to 1 week at −20°C.
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The molecular weight of Fluo-3 ester is 1130 g/mol. The concentrations of each indicator necessary must be determined empirically, focusing on matching the brightness of indicator 1 and Fura Red.
Fura Red AM, 10 mg/ml Dissolve Fura Red acetoxymethyl ester (Fura Red AM; Life Technologies, cat. no. F-3020) at 10 mg/ml in dimethyl sulfoxide (DMSO) and store in a desiccator in the dark up to 1 week at −20°C. The molecular weight of Fura Red ester is 945 g/mol.
Indo-1 AM (similar for Indo-5 F AM and Mag-Indo-1 AM), 2 mg/ml Dissolve Indo-1 acetoxymethyl ester (Indo-1 AM; Life Technologies cat. no. I-1223) at 2 mg/ml in dimethyl sulfoxide (DMSO) and store in a desiccator in the dark up to several months at −20°C. For a 50 μg aliquot of Indo-1 AM, add 25 μl DMSO. The molecular weight of Indo-1 ester is 1009.9 g/mol; a 2 mg/ml stock is almost exactly 2 mM.
Ionomycin, 1 mg/ml Dissolve at 1 mg/ml ionomycin (Millipore, cat. no. 407950) in dimethyl sulfoxide (DMSO) or 100% (v/v) ethanol. Use of DMSO minimizes evaporation of solvent and consequent concentration of ionomycin. Store up to 1 year at 4°C. Probenecid, 100 mM Life Technologies’ water-soluble probenecid (cat. no. P36400) removes the necessity for 1 M NaOH to dissolve it. The contents of one vial of 77 mg water-soluble probenecid and can be dissolved in 2.5 ml HBSS buffer for 100 mM final stock concentration. Otherwise, probenecid is relatively insoluble in aqueous solution, unless alkalinized and must be dissolved in water, adding 1 M NaOH until dissolved. The pH should be 9 to 10. COMMENTARY Background Information In their resting state, eukaryotic cells maintain an internal calcium ion concentration that is far below that of the extracellular environment. Ionized calcium has an important role as a mediator of transmembrane signal transduction, and elevations in intracellular ionized calcium concentration ([Ca2+ ]i ) regulate diverse cellular processes (Berridge, 2012). Thus, measurement of [Ca2+ ]i in living cells is of considerable interest to investigators in immunology and cell biology. Until 1980, measurement of [Ca2+ ]i was restricted to large invertebrate cells where the use of microelectrodes was possible. Subsequently, bioluminescent indicators such as aequorin, a calcium-activated photoprotein, were described, but were limited in their application by the necessity of loading cells by microinjection or other forms of membrane disruption (Blinks et al., 1982; Cobbold and Rink, 1987). Tsien and colleagues (Tsien et al., 1982) developed Quin-2, making it possible for the first
time to measure [Ca2+ ]i in virtually any population of cells. Unfortunately, Quin-2 has a relatively low extinction coefficient and quantum yield, and this, in conjunction with the fact that it does not have useful ratiometric properties, made the detection of calcium responses in single cells impractical. A second family of dyes was developed with the invention of Fura-2 and Indo-1 (Grynkiewicz et al., 1985), and it became possible to measure [Ca2+ ]i in single cells of almost any type. Fura-2 is best suited for applications involving fluorescence microscopy with digital image analysis, while Indo-1, due to its unique fluorescence properties, is best suited for flow cytometry. With Indo-1, there is a 6fold increase in signal when cells change from basal levels of [Ca2+ ]i to saturating amounts of [Ca2+ ]i . In addition, the ratio of Indo-1 signals allows measurement of [Ca2+ ]i that is independent of cell size or brightness. Cells loaded with Indo-1 exhibit a 3-fold range in brightness, and after measuring the ratio of
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Indo-1 calcium-bound to calcium-unbound at the proper wavelengths, the cell-to-cell variation in the signal from resting cells has a coefficient of variation of only 5% to 10% (Rabinovitch et al., 1986). Thus, with Indo-1 it is possible to discriminate responses of small subpopulations of cells within a larger population of non-responding cells. Flow cytometric cell conjugate assays have also been developed so that the calcium signals that occur during antigen presentation or target cell recognition can be studied using Indo-1 (Abe et al., 1992; Alexander et al., 1992; Van Graft et al., 1993). Another advantage of Indo-1 is sensitivity of response. Results of artificial mixing experiments with Jurkat T leukemia and K562 myeloid leukemia cell lines indicated that subpopulations of cells with variant [Ca2+ ]i comprising
