Cytometry 13545-552 (1992)
0 1992 Wiley-Liss, Inc.
TECHNICAL NOTES
Method to Improve the Sensitivity of Flow Cytometric Membrane Potential Measurements in Mouse Spinal Cord Cells' Larry C. Seame# and Raul N. Mandler Cytometry (L.C.S.) and Department of Neurology (R.N.M.), Cancer Center, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131. Received October 1, 1991; accepted January 11, 1992
We have developed a technique to improve the sensitivity of relative membrane potential measurements in mouse spinal cord cells using the fluorescent, anionic, voltage sensitive dye, DiBa-C,(3) (Oxonol) and flow cytometry. In order to attribute cellular fluorescence primarily to membrane potential, signal variability due to cell sue and shape was reduced by dividing the log fluorescence signal from each cell by either its log forward angle light scatter or log side scatter signals. The use of these ratios in place of log ox-
Membrane excitability has been studied in rodent spinal cord cells with the anionic voltage sensitive dye DiBa-C,(3) (oxonol) and flow cytometry (3,4). Oxonol has been used in our laboratory to study the response of mouse spinal cord cells from the wobbler mouse (a motor neuron disease model) to excitatory amino acids, neurotoxins, and steroids. Cellular loading with oxonol is a function of membrane potential; a s cell depolarization occurs, dye loading increases, causing a n increase in fluorescence intensity (2). Fluorescence intensity thus indirectly reflects membrane potential. However, when determining fluorescence intensity, the flow cytometer measures the total amount of fluorescent dye in each cell rather than the concentration of fluorescent molecules. Therefore, when cell suspensions such a s mouse spinal cord cells, which are heterogeneous in size and shape, are analyzed, a positive correlation between size and fluorescence is obtained, independent of membrane potential differences. This correlation suggests that size and possibly cell shape and subcellular structure are additional components of the oxonol fluorescence signal (10). These additional
onol fluorescence reduced the coefficient of variation of the distributions while leaving the changes in mean fluorescence largely unaffected. Kolmogorov-Smirnov analysis of pre- vs. postkainate stimulation (an excitatory amino acid) showed improved sensitivity of the assay with the use of this ratio technique. 0 1992 Wiley-Liss,Inc.
Key terms: Fluorescence normalization, light scatter, oxonol
dependent variables increase the coefficient of variation (CV) of oxonol fluorescence signal and contribute to a reduced sensitivity to small changes in membrane potential. To improve the sensitivity of the flow cytometric analysis of membrane potential, it was necessary to narrow the fluorescence distribution within the population of cells. The problem of fluorescence variation due to unequal dye loading has been addressed for other flow cytometric measurements, such as Caf determination with Indo-1, by calculating a ratio of fluorescence emissions of a single dye a t two wavelengths (7). Emission ratio normalization is only appro+
'This work was supported by NIH Grant NS27698,BRS6 SO7 RR05583-24 and S06GMO8139-18to R.N.M. 'Address reprint requests to Larry Seamer, Cytometry, Cancer Center, University of New Mexico, 915 Camino de Salud NE, Albuquerque, NM 87131.
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SEAMERANDMANDLER
priate if the relative intensities of the ratioed emissions change as a function of constituent (e.g., Ca+ +) content. This method is not appropriate when measuring relative membrane potential using oxonol because the emission spectra of oxonol remains relatively unchanged as a result of membrane potential changes. As a n alternate approach, fluorescence distributions can be narrowed by normalizing the fluorescence emission for cell size, by dividing fluorescence intensity by size on a cell by cell basis. Previous reports have shown that linear forward angle light scatter (FALS) can be used to calculate the density of cell surface antigens from fluorescence measurements by normalizing cellular fluorescence of antibody stained cells to cell size using FALS as the denominator in the normalization ratio (5,121. Side scatter (SSC) intensity reflects a complex mixture of a particle's size, shape, and internal structure. Because one of the variables associated with SSC is cellular size, it may also be useful for normalizing fluorescence intensity. In the current study we show that the sensitivity of membrane potential measurements can be increased by narrowing the fluorescence distribution of oxonolstained cells by dividing each cells log fluorescence intensity by its size as measured by both log FALS and log SSC. However, the relationship of these ratio parameters to oxonol density is unclear due to the logarithmic amplification of the signals.
MATERIALS AND METHODS Cells Adult, normal, and wobbler mice, a model for motor neuron disease, were sacrificed and the spinal cords removed by dissection. The spinal cord cells were dissociated using a n enzymatic protocol, aliquoted and stained with the anionic, voltage sensitive DiBa-C,(3) (Molecular Probes, Eugene, Or) immediately prior to flow cytometric analysis (3). Flow Cytometry Flow cytometry was performed using a Coulter, EPICS 753 (Coulter Corporation, Hialeah FL). Oxonol was excited with 600 mw at 488 nm using a Coherent Inova 90-5 laser (Coherent, Palo Alto, CAI. SSC emission was taken with a 515 nm long pass dichroic filter at 45" to the fluorescence light path. Oxonol fluorescence emission was measured through a second 515 nm long pass, laser blocking filter, and a 525k25 nm band pass filter. List mode files were collected on the following parameters: log integrated oxonol fluorescence, log SSC, log FALS and time. Data were acquired for 30 s, acquisition was briefly interrupted while the appropriate concentration of the excitatory amino acid, kainate (Sigma, St. Louis, MO), was added. Acquisition was then resumed for a n additional 140 s. This resulted in an 7-s gap in the data acquisition while the kainate was being added.
-
I FALS
I ssc FIG.1. Shows the correlation of (A) log FALS (x-axis) vs. log oxonol fluorescence (y-axis), and (B)log SSC (x-axis) vs. log oxonol fluorescence (y-axis). These contour plots were derived from the 30-kM kainate sample, using normal mouse spinal cord cells.
Data Analysis List mode files were transferred from the cytometer to a n IBM-PC compatible Easy2 cytometry workstation (Coulter Corporation, Hialeah, FL). Using Coulter Gateway software, two additional parameters were calculated from those obtained at acquisition. These parameters were: FALS-ratio = (log oxonol fluorescence/ log FALS)*C1 SSC-ratio = flog oxonol fluorescenceilog SSC)*C2 C1 and C2 were constants empirically derived to place the mean channel of the ratio parameters in the lower third of the fluorescence histogram. An important consideration was the analysis of FALS and SSC correlation with oxonol fluorescence. Using software developed inhouse, correlation coefficients were calculated using log FALS vs. log oxonol and log SSC vs. log oxonol parameters from list mode files (see Fig. 1).
547
INCREASED SENSITIVITY TO FLUORESCENCE CHANGES
A
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CHANNEL FIG.2. Shows the contour plot (A) of time (x-axis) vs. raw oxonol fluorescence (y-axis) of the 30 p M kainate sample from normal mouse spinal cord cells. The arrows and vertical lines indicate the time gates for creating fluorescence histograms (B) of baseline (solid line) and stimulated (dashed line) cells. The bottom chart (C) shows the summation curves for the two histograms used in the KolmogorovSmirnov test.
Two-parameter dot-plots of time (x-axis) vs. log oxonol fluorescence (y-axis), and similarly, time vs. FALS-ratio and time vs. SSC-ratio were created from list mode data (see Figs. 2A, 3A, 4A). Prereaction
(baseline) and a postreaction (kainate stimulated) single parameter histograms were derived by time gating on the two-parameter dot-plots over time intervals shown in A (see Figs. 2B, 3B, 4B). The pre- and post-
548
SEAMER AND MANDLER
A
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CHANNEL FIG.3. Similar to Figure 2, showing the same data for the FALS normalized reaction.
reaction sets of single parameter histograms were then analyzed for population variance by CV calculation. The histograms were compared for fluorescence shift by mean channel change and normalized to 10,000 counts for Kolmogorov-Smirnov (K-S) analysis (see Figs. 2C, 3C, 4C) (1,131. Fluorescence and ratio CVs were calculated from all
events falling between channel 2 and 255 on a-256 channels scale.
RESULTS Figure 1shows bivariate dot-plots of log FALS vs. log oxonol fluorescence (Fig. 1A) and log SSC vs log oxonol fluorescence (Fig. lB), graphically demonstrating the
549
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CHANNEL FIG.4. Similar to Figure 2, showing the same data for the SSC normalized reaction.
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Table 1 Results of a Membrane Potential Concentration-response on Normal Mouse and Wobbler Mouse Spinal Cord Cells in Response to the Excitatory Amino Acid, Kainate"
Normal Kainate 50pm 40pm
30pm 20pm
10pm Wobbler Kainate 50pm 40pm 30pm 20pm 10pm
AVERAGE
Raw data Fals ratio SSC ratio Mean Mean Mean Mean %change CV D-value Mean % chanee CV D-value Mean %change CV D-value Baseline Stimulated Baseline Stimulated Baseline Stimulated Baseline Stimulated Baseline Stimulated
178.1 132.2 118.2 144.3 109.7 141.0 124.0 136.7 123.6 126.0
Baseline Stimulated Baseline Stimulated Baseline Stimulated Baseline Stimulated Baseline Stimulated
105.4 140.1 113.6 134.2 106.2 129.0 122.1 130.4 118.9 133.0
40.9 18.1 22.2 9.3 1.9
44.3 29.2 31.3 25.2 37.4 27.3 30.2 27.2 31.4 29.6
10.6
35.2 28.6 31.9 27.5 36.1 29.5 30.0 28.0 32.9 28.6
16.7
31.1
24.8 15.4 17.7 6.4
~
56.4 28.0 31.0 13.2 4.8
37.2 24.0 22.5 10.0 15.2 24.2
65.4 109.1 99.9 120.5 90.3 122.0 104.1 114.0 101.2 106.8
89.6 120.3 96.9 113.6 89.8 108.3 105.2 112.9 101.6 113.9
40.1 17.1 26.0 8.7 5.2
33.5 20.3 20.4 18.4 25.1 18.1 20.4 17.9 20.0 21.4
10.8
23.3 20.6 22.7 21.6 25.7 20.3 21.9 23.1 23.3 20.6
17.2
21.9
25.5 14.7 17.1 6.8
75.6 44.2
50.0 21.8 15.9
58.3 35.1 37.5 15.3 23.7 37.7
74.2 121.8 117.0 135.8 100.5 128.8 116.4 127.7 113.0 120.8
100.9 136.7 108.9 128.3 100.8 122.2 118.5 129.1 114.9 130.4
39.1 13.8 22.0 8.8 6.5
34.8 20.3 21.4 17.4 25.8 18.0 20.6 18.2 20.4 21.1
11.9
23.8 20.6 23.1 20.4 26.3 20.3 22.0 22.0 23.8 20.6
16.9
22
26.2 15.1 17.5 8.2
75.3 48.0 53.3 23.5 19.6
61.4 37.3 38.0 20.0 27.8 40.4
"Mean channel. Dercent change in mean channel, CV, and K-S D =value are given for baseline and stimulated cells. Raw data is log oxonol fluorescence.-
correlation of light scatter and oxonol fluorescence. baseline mean channel fluorescence using the followThe mean correlation coefficient for all samples in ing formula: these experiments was 0.71 for log FALS vs. log oxonol (STIMULATED-BASELINE)/BASELINE * 100 fluorescence, and 0.75 for log SSC vs. log oxonol fluoThe average change in mean fluorescence between rescence. To measure an improvement in sensitivity, mean baseline and stimulated cells for FALS normalized hisfluorescence, CV, and K-S D-value for the baseline vs. tograms was 22.9%,which was a change of + 0.5%from stimulated population of the raw, FALS normalized, the shift seen in raw data. The average change in mean and SSC normalized fluorescence distributions were fluorescence for SSC normalized histograms was 22.2% recorded. Table 1 details the results of a kainate con- and showed almost no difference compared t o the shift centration-response experiment on normal and wob- seen in raw data, changing by an average of only bler mouse spinal cord cells. Results from both normal -0.1%. However, the population variance as measured and wobbler mouse experiments were analyzed to- by CV of the baseline fluorescence improved from an gether to obtain averages ( n = 10). Relative differences average value of CV = 31.1%for the raw histograms to in response t o kainate between normal and wobbler CV = 21.9% in the FALS normalized histograms and mouse cells are beyond the scope of this study and will CV = 22.0%in the SSC normalized histograms. This translated into a narrowing of the distribution and a be reported elsewhere. The shift in mean fluorescence of baseline vs. kain- subsequent improvement in sensitivity indicated by an ate stimulated cells showed little change with respect increased K-S D-value. The K-S D-value increased by a to the raw oxonol fluorescence histograms, the FALS minimum of 34.0% and an average of 55.2% for the normalized histograms, or the SSC normalized histo- FALS normalized data and a minimum of 33.5% and an grams. Since the magnitude of a fluorescence change is average of 69.7% for the SSC normalized fluorescence. dependent on histogram position for linear fluores- The improvement of K-S D-value resulted in an incence histograms (ratioed data becomes linear), mean crease in the sensitivity of the assay in the kainate channel changes are reported as a percentage of the concentration-response experiment, extending the
INCREASED SENSITIVITY TO FLUORESCENCE CHANGES
551
slight underlying membrane hyperpolarization caused by the ethanol. Although this method showed improvement in the CV of the oxonol fluorescence distributions, those dis70 tributions remained relatively broad. We believe this is 60 due to the complex correlation between light scatter and cell size. Light scatter, measured by the flow cy50 tometer, is proportional to cell size (6,9) and is a consequence of a particle passing through a focused light 40 beam. With commercial flow cytometers, light scatter is typically measured at two angles off the axis of the 30 excitation light path. FALS is a measure of the amount of light deflected by a cell (or any particle) in a narrow 20 angle off axis of the light source, which is approximately 1" to 19". However, the specific range of mea10 surement is dependent on the model of flow cytometer used. FALS has been shown to be correlated with the 40uM 30uM 20uM 1duM size of a cell as measured by a combination of volume KAINATE and cross sectional diameter (6).However, FALS measurement is also sensitive to other cellular properties FIG.5. Shows the results of the dose response experiment for raw oxonol fluorescence (m), FALS normalized oxonol fluorescence (0) and such as refractive index and internal structure (6,8,9). SSC normalized oxonol fluorescence (+I. SSC is a measure of the amount of light reflected at 90" off axis of the excitatory light and has been shown to be correlated with cytoplasmic granularity minimum detectable response from 20 p,M kainate for (11)and total protein content (9) as well as cell size. the raw fluorescence data, to 10 pM kainate for the The ratio of logarithmically amplified signals has narsize-normalized data, using either FALS or SSC for rowed the CV of the distributions; however, the relanormalization (Fig. 5). tionship of the ratio to oxonol density is unclear. As controls for these experiments, 500 pl cell suspenIn conclusion, the use of oxonolAight scatter ratios sions were treated with 2 pl of 70% ethanol, which was increases the sensitivity of membrane depolarization the diluent for the kainate in these experiments. These measurements in heterogeneous population of mouse control samples maintained in average D-value of spinal cord cells. We are currently using this technique 7.0%, which indicates no difference. Furthermore, the to explore the relative excitability of normal and wobsmall fluorescence shifts measured were in the oppo- bler mouse spinal cord cells exposed to a variety of site direction from the kainate-stimulated cells. depolarizing agents. This technique may also be applied t o improve the sensitivity of flow cytometric meaDISCUSSION surements in other similar systems in which fluoresFluorescence shifts as measured by Kolmogorov- cence staining is correlated with cell size. Smirnov analysis became more easily detectable when LITERATURE CITED analyzing log fluorescencellog light scatter ratios. SSC 1. Ault KA: Detection of small numbers of monoclonal B lymphoratios gave slightly better improvement. These results cytes in the blood of patients with lymphoma. New Engl J Med are predicted from the higher correlation coefficients 300:1401-1405, 1979. when comparing fluorescence and SSC with fluores2. Bashford CL, Alder GM, Gray MA, Micklem KJ, Taylor CC, cence and FALS. Cellular total protein may be contribTurek ? and Pasternak CA: Oxonol dyes as monitors of membrane uting to SSC signals (91, therefore yielding a better potential: The effect of viruses and toxins on the plasma membrane potential of animal cells in monolayer culture and in susestimation of cell volume and dye loading than FALS. pension. 3 Cell Physiol 123:326-336, 1985. The use of ratios in the analysis did not significantly 3. Mandler RN, Schaffner AE, Novotny EA, Lang GD, Smith SV, alter the mean shift. However, the use of ratios imBarker JL: Electrical and chemical excitability appear one week proved the CV of the fluorescence distributions, resultbefore birth in the embryonic rat spinal cord. Brain Res 522: ing in the improvement seen in K-S D-values. 46-54, 1990. 4. Mandler RN,Schaffner AE, Novotny EA, Lange GD, and Barker These improvements in D-value increased the sensiJL Flow cytometric analysis of membrane potential in embryonic tivity of the concentration-response experiments showrat spinal cord cells. J Neurosci Meth 22203-213, 1988. ing detectable response down to 10 pM kainate with 5. Matsui Y,Staunton DE, Shapiro HM and Yunis EJ: Comparison either FALS or SSC normalized oxonol fluorescence, of MHC antigen expression on PHA- and MLC-induced T cell lines with that on T and B lymphoblastoid cell lines by cell cycle whereas the raw (nonnormalized) data showed redependency. Human Immunol 15:285-301,1986, sponse only down to 20 ~,LMkainate. Control samples 6. Melamed MR, Lindmo T, Mendelsohn ML: Flow Cytometry and remained within the range of nonsignificance when Sorting, 2nd ed. New York Wiley-Liss, 1990. normalized. The shift in mean channel of these controls 7. Rabinovitch P,June C, Grossman A, Ledbetter J: Heterogeneity decreased when compared to the baseline, indicating a among T cells in intracellular free calcium responses after mitoK
S TEST
80
-
552
SEAMERANDMANDLER
gen stimulation with PHA or anti-CD3. Simultaneous use of indo1and immunofluorescence with flow cytometry. J Immunoll37: 952-961,1986. 8. Salzman GJ, Wilder ME and Jett JH: Light scattering with stream in air flow systems. Histochem and Cytochem 27:264267,1979. 9. Shapiro HM: Practical Flow Cytometry. New York: Alan R. Liss, 1988. 10. Shapiro HM, Natale PJ: Estimation of membrane potentials of individual lymphocytes hy flow cytometry. PNAS 76:5728-5730, 1979. 11. Sklar LA, Oades ZG and Finney DA: Neutrophil degranulation
detected by right angle scattering: Spectroscopic methods suitable for simultaneous analysis of degranulation or shape change, elastase release, and cell aggregation, J Immunol 133:14831487, 1984. 12. Steinkamp JA, Kraemer PM: Flow microfluorometric studies of lectin binding to mammalian cells. 11. Estimation of the surface density of receptor sites by multiparameter analysis, J Cell Physiol 84:197-204, 1974. 13. Young IT: Proof without prejudice: Use of the KolmogorovSmirnov test for the analysis of histograms from flow systems and other sources. J Histochem Cytochem 25:935-941, 1977.
0 1992 Wiley-Liss, Inc.
TECHNICAL NOTES
Method to Improve the Sensitivity of Flow Cytometric Membrane Potential Measurements in Mouse Spinal Cord Cells' Larry C. Seame# and Raul N. Mandler Cytometry (L.C.S.) and Department of Neurology (R.N.M.), Cancer Center, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131. Received October 1, 1991; accepted January 11, 1992
We have developed a technique to improve the sensitivity of relative membrane potential measurements in mouse spinal cord cells using the fluorescent, anionic, voltage sensitive dye, DiBa-C,(3) (Oxonol) and flow cytometry. In order to attribute cellular fluorescence primarily to membrane potential, signal variability due to cell sue and shape was reduced by dividing the log fluorescence signal from each cell by either its log forward angle light scatter or log side scatter signals. The use of these ratios in place of log ox-
Membrane excitability has been studied in rodent spinal cord cells with the anionic voltage sensitive dye DiBa-C,(3) (oxonol) and flow cytometry (3,4). Oxonol has been used in our laboratory to study the response of mouse spinal cord cells from the wobbler mouse (a motor neuron disease model) to excitatory amino acids, neurotoxins, and steroids. Cellular loading with oxonol is a function of membrane potential; a s cell depolarization occurs, dye loading increases, causing a n increase in fluorescence intensity (2). Fluorescence intensity thus indirectly reflects membrane potential. However, when determining fluorescence intensity, the flow cytometer measures the total amount of fluorescent dye in each cell rather than the concentration of fluorescent molecules. Therefore, when cell suspensions such a s mouse spinal cord cells, which are heterogeneous in size and shape, are analyzed, a positive correlation between size and fluorescence is obtained, independent of membrane potential differences. This correlation suggests that size and possibly cell shape and subcellular structure are additional components of the oxonol fluorescence signal (10). These additional
onol fluorescence reduced the coefficient of variation of the distributions while leaving the changes in mean fluorescence largely unaffected. Kolmogorov-Smirnov analysis of pre- vs. postkainate stimulation (an excitatory amino acid) showed improved sensitivity of the assay with the use of this ratio technique. 0 1992 Wiley-Liss,Inc.
Key terms: Fluorescence normalization, light scatter, oxonol
dependent variables increase the coefficient of variation (CV) of oxonol fluorescence signal and contribute to a reduced sensitivity to small changes in membrane potential. To improve the sensitivity of the flow cytometric analysis of membrane potential, it was necessary to narrow the fluorescence distribution within the population of cells. The problem of fluorescence variation due to unequal dye loading has been addressed for other flow cytometric measurements, such as Caf determination with Indo-1, by calculating a ratio of fluorescence emissions of a single dye a t two wavelengths (7). Emission ratio normalization is only appro+
'This work was supported by NIH Grant NS27698,BRS6 SO7 RR05583-24 and S06GMO8139-18to R.N.M. 'Address reprint requests to Larry Seamer, Cytometry, Cancer Center, University of New Mexico, 915 Camino de Salud NE, Albuquerque, NM 87131.
546
SEAMERANDMANDLER
priate if the relative intensities of the ratioed emissions change as a function of constituent (e.g., Ca+ +) content. This method is not appropriate when measuring relative membrane potential using oxonol because the emission spectra of oxonol remains relatively unchanged as a result of membrane potential changes. As a n alternate approach, fluorescence distributions can be narrowed by normalizing the fluorescence emission for cell size, by dividing fluorescence intensity by size on a cell by cell basis. Previous reports have shown that linear forward angle light scatter (FALS) can be used to calculate the density of cell surface antigens from fluorescence measurements by normalizing cellular fluorescence of antibody stained cells to cell size using FALS as the denominator in the normalization ratio (5,121. Side scatter (SSC) intensity reflects a complex mixture of a particle's size, shape, and internal structure. Because one of the variables associated with SSC is cellular size, it may also be useful for normalizing fluorescence intensity. In the current study we show that the sensitivity of membrane potential measurements can be increased by narrowing the fluorescence distribution of oxonolstained cells by dividing each cells log fluorescence intensity by its size as measured by both log FALS and log SSC. However, the relationship of these ratio parameters to oxonol density is unclear due to the logarithmic amplification of the signals.
MATERIALS AND METHODS Cells Adult, normal, and wobbler mice, a model for motor neuron disease, were sacrificed and the spinal cords removed by dissection. The spinal cord cells were dissociated using a n enzymatic protocol, aliquoted and stained with the anionic, voltage sensitive DiBa-C,(3) (Molecular Probes, Eugene, Or) immediately prior to flow cytometric analysis (3). Flow Cytometry Flow cytometry was performed using a Coulter, EPICS 753 (Coulter Corporation, Hialeah FL). Oxonol was excited with 600 mw at 488 nm using a Coherent Inova 90-5 laser (Coherent, Palo Alto, CAI. SSC emission was taken with a 515 nm long pass dichroic filter at 45" to the fluorescence light path. Oxonol fluorescence emission was measured through a second 515 nm long pass, laser blocking filter, and a 525k25 nm band pass filter. List mode files were collected on the following parameters: log integrated oxonol fluorescence, log SSC, log FALS and time. Data were acquired for 30 s, acquisition was briefly interrupted while the appropriate concentration of the excitatory amino acid, kainate (Sigma, St. Louis, MO), was added. Acquisition was then resumed for a n additional 140 s. This resulted in an 7-s gap in the data acquisition while the kainate was being added.
-
I FALS
I ssc FIG.1. Shows the correlation of (A) log FALS (x-axis) vs. log oxonol fluorescence (y-axis), and (B)log SSC (x-axis) vs. log oxonol fluorescence (y-axis). These contour plots were derived from the 30-kM kainate sample, using normal mouse spinal cord cells.
Data Analysis List mode files were transferred from the cytometer to a n IBM-PC compatible Easy2 cytometry workstation (Coulter Corporation, Hialeah, FL). Using Coulter Gateway software, two additional parameters were calculated from those obtained at acquisition. These parameters were: FALS-ratio = (log oxonol fluorescence/ log FALS)*C1 SSC-ratio = flog oxonol fluorescenceilog SSC)*C2 C1 and C2 were constants empirically derived to place the mean channel of the ratio parameters in the lower third of the fluorescence histogram. An important consideration was the analysis of FALS and SSC correlation with oxonol fluorescence. Using software developed inhouse, correlation coefficients were calculated using log FALS vs. log oxonol and log SSC vs. log oxonol parameters from list mode files (see Fig. 1).
547
INCREASED SENSITIVITY TO FLUORESCENCE CHANGES
A
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CHANNEL FIG.2. Shows the contour plot (A) of time (x-axis) vs. raw oxonol fluorescence (y-axis) of the 30 p M kainate sample from normal mouse spinal cord cells. The arrows and vertical lines indicate the time gates for creating fluorescence histograms (B) of baseline (solid line) and stimulated (dashed line) cells. The bottom chart (C) shows the summation curves for the two histograms used in the KolmogorovSmirnov test.
Two-parameter dot-plots of time (x-axis) vs. log oxonol fluorescence (y-axis), and similarly, time vs. FALS-ratio and time vs. SSC-ratio were created from list mode data (see Figs. 2A, 3A, 4A). Prereaction
(baseline) and a postreaction (kainate stimulated) single parameter histograms were derived by time gating on the two-parameter dot-plots over time intervals shown in A (see Figs. 2B, 3B, 4B). The pre- and post-
548
SEAMER AND MANDLER
A
B I'
BASt'
U
':
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CHANNEL NUMBER FALS RATIO
2oool
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cn
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8000-
w
6000-
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21
41
61
81 101 121 141 161 181 201 221 241
CHANNEL FIG.3. Similar to Figure 2, showing the same data for the FALS normalized reaction.
reaction sets of single parameter histograms were then analyzed for population variance by CV calculation. The histograms were compared for fluorescence shift by mean channel change and normalized to 10,000 counts for Kolmogorov-Smirnov (K-S) analysis (see Figs. 2C, 3C, 4C) (1,131. Fluorescence and ratio CVs were calculated from all
events falling between channel 2 and 255 on a-256 channels scale.
RESULTS Figure 1shows bivariate dot-plots of log FALS vs. log oxonol fluorescence (Fig. 1A) and log SSC vs log oxonol fluorescence (Fig. lB), graphically demonstrating the
549
INCREASED SENSITIVITY TO FLUORESCENCE CHANGES
TI ME
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CHANNEL FIG.4. Similar to Figure 2, showing the same data for the SSC normalized reaction.
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Table 1 Results of a Membrane Potential Concentration-response on Normal Mouse and Wobbler Mouse Spinal Cord Cells in Response to the Excitatory Amino Acid, Kainate"
Normal Kainate 50pm 40pm
30pm 20pm
10pm Wobbler Kainate 50pm 40pm 30pm 20pm 10pm
AVERAGE
Raw data Fals ratio SSC ratio Mean Mean Mean Mean %change CV D-value Mean % chanee CV D-value Mean %change CV D-value Baseline Stimulated Baseline Stimulated Baseline Stimulated Baseline Stimulated Baseline Stimulated
178.1 132.2 118.2 144.3 109.7 141.0 124.0 136.7 123.6 126.0
Baseline Stimulated Baseline Stimulated Baseline Stimulated Baseline Stimulated Baseline Stimulated
105.4 140.1 113.6 134.2 106.2 129.0 122.1 130.4 118.9 133.0
40.9 18.1 22.2 9.3 1.9
44.3 29.2 31.3 25.2 37.4 27.3 30.2 27.2 31.4 29.6
10.6
35.2 28.6 31.9 27.5 36.1 29.5 30.0 28.0 32.9 28.6
16.7
31.1
24.8 15.4 17.7 6.4
~
56.4 28.0 31.0 13.2 4.8
37.2 24.0 22.5 10.0 15.2 24.2
65.4 109.1 99.9 120.5 90.3 122.0 104.1 114.0 101.2 106.8
89.6 120.3 96.9 113.6 89.8 108.3 105.2 112.9 101.6 113.9
40.1 17.1 26.0 8.7 5.2
33.5 20.3 20.4 18.4 25.1 18.1 20.4 17.9 20.0 21.4
10.8
23.3 20.6 22.7 21.6 25.7 20.3 21.9 23.1 23.3 20.6
17.2
21.9
25.5 14.7 17.1 6.8
75.6 44.2
50.0 21.8 15.9
58.3 35.1 37.5 15.3 23.7 37.7
74.2 121.8 117.0 135.8 100.5 128.8 116.4 127.7 113.0 120.8
100.9 136.7 108.9 128.3 100.8 122.2 118.5 129.1 114.9 130.4
39.1 13.8 22.0 8.8 6.5
34.8 20.3 21.4 17.4 25.8 18.0 20.6 18.2 20.4 21.1
11.9
23.8 20.6 23.1 20.4 26.3 20.3 22.0 22.0 23.8 20.6
16.9
22
26.2 15.1 17.5 8.2
75.3 48.0 53.3 23.5 19.6
61.4 37.3 38.0 20.0 27.8 40.4
"Mean channel. Dercent change in mean channel, CV, and K-S D =value are given for baseline and stimulated cells. Raw data is log oxonol fluorescence.-
correlation of light scatter and oxonol fluorescence. baseline mean channel fluorescence using the followThe mean correlation coefficient for all samples in ing formula: these experiments was 0.71 for log FALS vs. log oxonol (STIMULATED-BASELINE)/BASELINE * 100 fluorescence, and 0.75 for log SSC vs. log oxonol fluoThe average change in mean fluorescence between rescence. To measure an improvement in sensitivity, mean baseline and stimulated cells for FALS normalized hisfluorescence, CV, and K-S D-value for the baseline vs. tograms was 22.9%,which was a change of + 0.5%from stimulated population of the raw, FALS normalized, the shift seen in raw data. The average change in mean and SSC normalized fluorescence distributions were fluorescence for SSC normalized histograms was 22.2% recorded. Table 1 details the results of a kainate con- and showed almost no difference compared t o the shift centration-response experiment on normal and wob- seen in raw data, changing by an average of only bler mouse spinal cord cells. Results from both normal -0.1%. However, the population variance as measured and wobbler mouse experiments were analyzed to- by CV of the baseline fluorescence improved from an gether to obtain averages ( n = 10). Relative differences average value of CV = 31.1%for the raw histograms to in response t o kainate between normal and wobbler CV = 21.9% in the FALS normalized histograms and mouse cells are beyond the scope of this study and will CV = 22.0%in the SSC normalized histograms. This translated into a narrowing of the distribution and a be reported elsewhere. The shift in mean fluorescence of baseline vs. kain- subsequent improvement in sensitivity indicated by an ate stimulated cells showed little change with respect increased K-S D-value. The K-S D-value increased by a to the raw oxonol fluorescence histograms, the FALS minimum of 34.0% and an average of 55.2% for the normalized histograms, or the SSC normalized histo- FALS normalized data and a minimum of 33.5% and an grams. Since the magnitude of a fluorescence change is average of 69.7% for the SSC normalized fluorescence. dependent on histogram position for linear fluores- The improvement of K-S D-value resulted in an incence histograms (ratioed data becomes linear), mean crease in the sensitivity of the assay in the kainate channel changes are reported as a percentage of the concentration-response experiment, extending the
INCREASED SENSITIVITY TO FLUORESCENCE CHANGES
551
slight underlying membrane hyperpolarization caused by the ethanol. Although this method showed improvement in the CV of the oxonol fluorescence distributions, those dis70 tributions remained relatively broad. We believe this is 60 due to the complex correlation between light scatter and cell size. Light scatter, measured by the flow cy50 tometer, is proportional to cell size (6,9) and is a consequence of a particle passing through a focused light 40 beam. With commercial flow cytometers, light scatter is typically measured at two angles off the axis of the 30 excitation light path. FALS is a measure of the amount of light deflected by a cell (or any particle) in a narrow 20 angle off axis of the light source, which is approximately 1" to 19". However, the specific range of mea10 surement is dependent on the model of flow cytometer used. FALS has been shown to be correlated with the 40uM 30uM 20uM 1duM size of a cell as measured by a combination of volume KAINATE and cross sectional diameter (6).However, FALS measurement is also sensitive to other cellular properties FIG.5. Shows the results of the dose response experiment for raw oxonol fluorescence (m), FALS normalized oxonol fluorescence (0) and such as refractive index and internal structure (6,8,9). SSC normalized oxonol fluorescence (+I. SSC is a measure of the amount of light reflected at 90" off axis of the excitatory light and has been shown to be correlated with cytoplasmic granularity minimum detectable response from 20 p,M kainate for (11)and total protein content (9) as well as cell size. the raw fluorescence data, to 10 pM kainate for the The ratio of logarithmically amplified signals has narsize-normalized data, using either FALS or SSC for rowed the CV of the distributions; however, the relanormalization (Fig. 5). tionship of the ratio to oxonol density is unclear. As controls for these experiments, 500 pl cell suspenIn conclusion, the use of oxonolAight scatter ratios sions were treated with 2 pl of 70% ethanol, which was increases the sensitivity of membrane depolarization the diluent for the kainate in these experiments. These measurements in heterogeneous population of mouse control samples maintained in average D-value of spinal cord cells. We are currently using this technique 7.0%, which indicates no difference. Furthermore, the to explore the relative excitability of normal and wobsmall fluorescence shifts measured were in the oppo- bler mouse spinal cord cells exposed to a variety of site direction from the kainate-stimulated cells. depolarizing agents. This technique may also be applied t o improve the sensitivity of flow cytometric meaDISCUSSION surements in other similar systems in which fluoresFluorescence shifts as measured by Kolmogorov- cence staining is correlated with cell size. Smirnov analysis became more easily detectable when LITERATURE CITED analyzing log fluorescencellog light scatter ratios. SSC 1. Ault KA: Detection of small numbers of monoclonal B lymphoratios gave slightly better improvement. These results cytes in the blood of patients with lymphoma. New Engl J Med are predicted from the higher correlation coefficients 300:1401-1405, 1979. when comparing fluorescence and SSC with fluores2. Bashford CL, Alder GM, Gray MA, Micklem KJ, Taylor CC, cence and FALS. Cellular total protein may be contribTurek ? and Pasternak CA: Oxonol dyes as monitors of membrane uting to SSC signals (91, therefore yielding a better potential: The effect of viruses and toxins on the plasma membrane potential of animal cells in monolayer culture and in susestimation of cell volume and dye loading than FALS. pension. 3 Cell Physiol 123:326-336, 1985. The use of ratios in the analysis did not significantly 3. Mandler RN, Schaffner AE, Novotny EA, Lang GD, Smith SV, alter the mean shift. However, the use of ratios imBarker JL: Electrical and chemical excitability appear one week proved the CV of the fluorescence distributions, resultbefore birth in the embryonic rat spinal cord. Brain Res 522: ing in the improvement seen in K-S D-values. 46-54, 1990. 4. Mandler RN,Schaffner AE, Novotny EA, Lange GD, and Barker These improvements in D-value increased the sensiJL Flow cytometric analysis of membrane potential in embryonic tivity of the concentration-response experiments showrat spinal cord cells. J Neurosci Meth 22203-213, 1988. ing detectable response down to 10 pM kainate with 5. Matsui Y,Staunton DE, Shapiro HM and Yunis EJ: Comparison either FALS or SSC normalized oxonol fluorescence, of MHC antigen expression on PHA- and MLC-induced T cell lines with that on T and B lymphoblastoid cell lines by cell cycle whereas the raw (nonnormalized) data showed redependency. Human Immunol 15:285-301,1986, sponse only down to 20 ~,LMkainate. Control samples 6. Melamed MR, Lindmo T, Mendelsohn ML: Flow Cytometry and remained within the range of nonsignificance when Sorting, 2nd ed. New York Wiley-Liss, 1990. normalized. The shift in mean channel of these controls 7. Rabinovitch P,June C, Grossman A, Ledbetter J: Heterogeneity decreased when compared to the baseline, indicating a among T cells in intracellular free calcium responses after mitoK
S TEST
80
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gen stimulation with PHA or anti-CD3. Simultaneous use of indo1and immunofluorescence with flow cytometry. J Immunoll37: 952-961,1986. 8. Salzman GJ, Wilder ME and Jett JH: Light scattering with stream in air flow systems. Histochem and Cytochem 27:264267,1979. 9. Shapiro HM: Practical Flow Cytometry. New York: Alan R. Liss, 1988. 10. Shapiro HM, Natale PJ: Estimation of membrane potentials of individual lymphocytes hy flow cytometry. PNAS 76:5728-5730, 1979. 11. Sklar LA, Oades ZG and Finney DA: Neutrophil degranulation
detected by right angle scattering: Spectroscopic methods suitable for simultaneous analysis of degranulation or shape change, elastase release, and cell aggregation, J Immunol 133:14831487, 1984. 12. Steinkamp JA, Kraemer PM: Flow microfluorometric studies of lectin binding to mammalian cells. 11. Estimation of the surface density of receptor sites by multiparameter analysis, J Cell Physiol 84:197-204, 1974. 13. Young IT: Proof without prejudice: Use of the KolmogorovSmirnov test for the analysis of histograms from flow systems and other sources. J Histochem Cytochem 25:935-941, 1977.
