Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on December 15, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Tracking Individual Intracellular Proteins Using Quantum Dots Sébastien Courty and Maxime Dahan
Single-molecule detection of quantum dot (QD)-tagged proteins located in the cytoplasm or the nucleus presents a significant challenge in live-cell imaging. First, QDs must enter the cell cytoplasm and reach their molecular target but still preserve cell integrity. Second, the fluorescence of individual QDs must be detected in a noisy environment and distinguished from the autofluorescence of intracellular compartments and organelles. Finally, molecular motion in the cytosol is likely to be threedimensional, compared to two-dimensional diffusion in the membrane. In this protocol, streptavidincoated QDs (QD-SAVs) are coupled with biotinylated proteins (ideally in a 1:1 molar ratio) in hypertonic medium. The coupled reaction product (QD-P) is then added to live cells (e.g., mammalian HeLa cells) using a cell-loading technique based on the osmotic lysis of pinocytic vesicles. The osmotic lysis of pinocytic vesicles in hypotonic solution does not alter the viability of cultured cells and does not result in lysosomal enzyme release. By comparison with other internalization techniques, such as microinjection, this method is much simpler and more reproducible because all of the cells are simultaneously loaded under the same conditions. It can provide quantitative information on the movement of intracellular biomolecules, enhancing our understanding of complex biological processes such as signal transduction, cell division, or motility.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol.
Reagents
Biotinylated primary antibody (monoclonal or polyclonal against protein of interest) Cultured cells on glass coverslips The incubation volumes indicated below correspond to a circular coverslip with an 18-mm diameter.
Dulbecco’s minimal essential medium (DMEM) Fetal bovine serum (FBS) HEPES (1 M, pH 7.4) Hypotonic lysis medium Prepare by combining DMEM medium, without serum, and H2O in a 6:4 ratio.
Influx pinocytic cell-loading reagent (Life Technologies I-14402) The Influx cell-loading technique for water-soluble compounds is based on the osmotic lysis of pinocytic vesicles (Okada and Rechsteiner 1982). Compounds to be loaded are mixed at high concentration in a hypertonic medium (prepared from this reagent), allowing the material to be carried into the cells via pinocytic vesicles. The cells are then transferred to a hypotonic medium, which results in the release of trapped material from the vesicles into the cells.
Adapted from Single-Molecule Techniques (ed. Selvin and Ha). CSHL Press, Cold Spring Harbor, NY, USA, 2008. © 2013 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078238
1076
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on December 15, 2014 - Published by Cold Spring Harbor Laboratory Press
Tracking Intracellular Proteins Using QDs
MEM imaging medium (e.g., Life Technologies) This live-cell visualization medium is supplemented with 2 mM glutamine, 1% penicillin–streptomycin, and 10% FBS.
Streptavidin-coated quantum dots (QD-SAVs) (Life Technologies) Equipment
Attofluor cell chamber (Life Technologies A-7816) Autoclave Coverslips (circular; 18-mm diameter; sterile) Culture dish Forceps (sterile) Parafilm Plastic box Staining jar (or mini-rack) Thermobloc Tissues (sterile) Tubes (sterile; 15 mL and 50 mL; Falcon) Vortex mixer Water baths (preset to 37˚C and 80˚C) METHOD Preparation of Hypertonic Loading Medium Containing QD-Ps
1. Prewarm 5 mL of DMEM medium, without serum, to 37˚C.
2. Melt the polyethylene glycol (waxy solid on top of sucrose crystals) contained in the tube of Influx Pinocytic Cell-Loading Reagent by heating the tube in hot water (80˚C) for 2 min. 3. Remove the cap of the tube and quickly add 4.7 mL of DMEM medium, without serum, at 37˚C.
4. Replace the cap and vortex the tube vigorously to completely dissolve the sucrose crystals.
5. Once the medium is homogeneous, add 250 µL of FBS and 50 µL of 1 M HEPES buffer at pH 7.4 (or other suitable buffer, depending on the cell type). Replace the cap and vortex the tube for a few minutes. The hypertonic medium can be stored at 4˚C for up to several weeks. Keep the tube capped until immediately before use. Just before the experiments, open the tube in a sterile environment (e.g., close to a flame) to keep the hypertonic medium sterile. Check the color of the medium, which should be red if no contamination is found, assuming that the pH indicator is phenol red.
6. Immediately before use, prepare the QD-Ps (coupled reaction product) by incubating QD-SAVs (at a final concentration of 1 nM) with biotinylated protein (ideally in a 1:1 molar ratio) in the hypertonic medium. Internalization of QD-Ps into Live Cultured Cells The cell-loading technique used here is based on the procedure described by Okada and Rechsteiner (1982).
7. Plate cultured cells at a density of 1.5 × 105 cells/cm2 on 18-mm glass coverslips that have been maintained in DMEM medium containing 10% FBS at 37˚C in a 5% CO2 atmosphere.
8. Prewarm 100 µL each of hypertonic medium containing QD-Ps, hypotonic lysis medium, and MEM imaging medium to 37˚C (assuming that this is the ideal temperature for the cell type). 9. Use sterile forceps to collect a coverslip from the culture dish.
10. Remove the excess medium by touching the edge of the coverslip to a sterile tissue. Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078238
1077
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on December 15, 2014 - Published by Cold Spring Harbor Laboratory Press
S. Courty and M. Dahan
FIGURE 1. Detection of single intracellular QD-tagged proteins. (A) Bright-field image of a cultured HeLa cell. (B) Fluorescence image of individual QDs internalized in the cytoplasm. (C,D) In the case of small fluorescent dextran molecules internalized with the same protocol, the molecules diffuse rapidly in the cytoplasm, leading to a uniform staining.
11. Place the coverslip cell-side up on a piece of parafilm (5 × 5 cm) heated at 37˚C with a thermobloc. 12. Quickly but gently deposit 100 µL of hypertonic medium containing QD-Ps on the coverslip boundary. 13. Incubate the cells for 10 min at 37˚C in the hypertonic medium along with QD-Ps. Cover the coverslip with a plastic box to avoid evaporation. See Troubleshooting.
14. Repeat Step 10. 15. Place the coverslip vertically in a staining jar (alternatively, a mini-rack can be used), filled with at least 7 mL of prewarmed hypotonic lysis medium, for exactly 2 min. After osmotic lysis of pinocytic vesicles, induced by incubating the cells in a hypotonic lysis medium, QD-Ps are released homogeneously in the cell cytosol. See Troubleshooting.
16. Repeat Step 10. 17. Place the coverslip in the cell chamber filled with MEM imaging medium prewarmed to 37˚C.
18. Incubate the cells for 10 min at 37˚C to promote recovery.
19. Image the cells, using appropriate settings.
Figure 1 shows an example of detection of single intracellular QD-tagged proteins. See Troubleshooting.
TROUBLESHOOTING Problem (Step 13): Cells do not adhere to coverslips. Solution: For nonadhesive cells (e.g., Drosophila cells), use the following procedure for coating
coverslips with poly-L-lysine (this may be important for Step 15 in particular because the viscosity of the hypertonic medium may remove cells from the glass coverslip): 1. Autoclave the coverslips to be used. 2. Rinse the coverslips with phosphate-buffered saline (PBS) to remove any debris and to uniformly wet their surfaces. 3. Cover the surface of the coverslip with 200 µL of a solution of poly-L-lysine (1 mg/mL; SigmaAldrich). 1078
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078238
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on December 15, 2014 - Published by Cold Spring Harbor Laboratory Press
Tracking Intracellular Proteins Using QDs
4. Incubate the coverslips in poly-L-lysine for at least 1 h. 5. Aspirate and allow the coverslips to dry completely (overnight if necessary). 6. Gently wash the coverslips (at least five times) with PBS to remove any excess poly-L-lysine, which appears to be very toxic to cells. 7. Store poly-L-lysine-coated coverslips in PBS at 4˚C for up to 2 wk in a clean, dry place to avoid dust and humidity until needed. Problem (Step 15): Cell viability is low. Solution: Reduce the exposure time in the hypotonic lysis medium; too long an exposure may result in
blebbing of the cell membrane. Problem (Step 19): There is cell autofluorescence. Solution: Selecting the right QD emission wavelength is important for overcoming cell autofluor-
escence. Cells show autofluorescence composed of at least four distinct excitation and emission maxima from endogenous metabolites: the tryptophan peak (290-nm excitation, 330-nm emission), the NAD(P)H peak (350-nm excitation, 450-nm emission), the riboflavin (FAD) peak (450-nm excitation, 530-nm emission), and a yet unidentified peak (500-nm excitation, 530-nm emission) (Heintzelman et al. 2000). Note also that autofluorescence varies greatly in living cells. For example, autofluorescence is low in freshly prepared cells and increases with time during culture until a plateau is reached. To avoid any overlap with the cell autofluorescence, we thus recommend using QDs emitting in the red range at 655 nm. The main difficulty in tracking single QDs inside living cells comes from their three-dimensional motion, which might lead to defocusing of the fluorescence spots. Unfortunately, no general method currently exists to overcome this problem. We thus recommend, whenever possible, to focus on a part where the cells are relatively flat on the coverslip. RELATED INFORMATION
This protocol has been successfully implemented for tracking single biotinylated-kinesin molecules coupled to QD-SAVs and internalized within living cultured cells (Courty et al. 2006). Single-particle tracking has also proven to be a powerful method for deciphering the molecular organization of the plasma membrane (see Tracking Individual Membrane Proteins Using Quantum Dots [Courty and Dahan 2013a]). For a discussion of practical issues related to the detection of individual nanoparticles by wide-field fluorescence microscopy, see Ultrasensitive Imaging in Live Cells Using Fluorescent Quantum Dots (Courty and Dahan 2013b). REFERENCES Courty S, Luccardini C, Bellaiche Y, Cappello G, Dahan M. 2006. Tracking individual kinesin motors in living cells using single quantum-dot imaging. Nano Lett 6: 1491–1495. Courty S, Dahan M. 2013a. Tracking individual membrane proteins using quantum dots. Cold Spring Harb Protoc doi: 10.1101/pdb.prot078196. Courty S, Dahan M. 2013b. Ultrasensitive imaging in live cells using fluorescent quantum dots. Cold Spring Harb Protoc doi: 10.1101/pdb. top078220.
Heintzelman DL, Lotan R, Richards-Kortum RR. 2000. Characterization of the autofluorescence of polymorphonuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia. Photochem Photobiol 71: 327–332. Okada CY, Rechsteiner M. 1982. Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell 29: 33–41.
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078238
1079
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on December 15, 2014 - Published by Cold Spring Harbor Laboratory Press
Tracking Individual Intracellular Proteins Using Quantum Dots Sébastien Courty and Maxime Dahan Cold Spring Harb Protoc; doi: 10.1101/pdb.prot078238 Email Alerting Service Subject Categories
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To subscribe to Cold Spring Harbor Protocols go to:
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© 2013 Cold Spring Harbor Laboratory Press
Protocol
Tracking Individual Intracellular Proteins Using Quantum Dots Sébastien Courty and Maxime Dahan
Single-molecule detection of quantum dot (QD)-tagged proteins located in the cytoplasm or the nucleus presents a significant challenge in live-cell imaging. First, QDs must enter the cell cytoplasm and reach their molecular target but still preserve cell integrity. Second, the fluorescence of individual QDs must be detected in a noisy environment and distinguished from the autofluorescence of intracellular compartments and organelles. Finally, molecular motion in the cytosol is likely to be threedimensional, compared to two-dimensional diffusion in the membrane. In this protocol, streptavidincoated QDs (QD-SAVs) are coupled with biotinylated proteins (ideally in a 1:1 molar ratio) in hypertonic medium. The coupled reaction product (QD-P) is then added to live cells (e.g., mammalian HeLa cells) using a cell-loading technique based on the osmotic lysis of pinocytic vesicles. The osmotic lysis of pinocytic vesicles in hypotonic solution does not alter the viability of cultured cells and does not result in lysosomal enzyme release. By comparison with other internalization techniques, such as microinjection, this method is much simpler and more reproducible because all of the cells are simultaneously loaded under the same conditions. It can provide quantitative information on the movement of intracellular biomolecules, enhancing our understanding of complex biological processes such as signal transduction, cell division, or motility.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol.
Reagents
Biotinylated primary antibody (monoclonal or polyclonal against protein of interest) Cultured cells on glass coverslips The incubation volumes indicated below correspond to a circular coverslip with an 18-mm diameter.
Dulbecco’s minimal essential medium (DMEM) Fetal bovine serum (FBS) HEPES (1 M, pH 7.4) Hypotonic lysis medium Prepare by combining DMEM medium, without serum, and H2O in a 6:4 ratio.
Influx pinocytic cell-loading reagent (Life Technologies I-14402) The Influx cell-loading technique for water-soluble compounds is based on the osmotic lysis of pinocytic vesicles (Okada and Rechsteiner 1982). Compounds to be loaded are mixed at high concentration in a hypertonic medium (prepared from this reagent), allowing the material to be carried into the cells via pinocytic vesicles. The cells are then transferred to a hypotonic medium, which results in the release of trapped material from the vesicles into the cells.
Adapted from Single-Molecule Techniques (ed. Selvin and Ha). CSHL Press, Cold Spring Harbor, NY, USA, 2008. © 2013 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078238
1076
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on December 15, 2014 - Published by Cold Spring Harbor Laboratory Press
Tracking Intracellular Proteins Using QDs
MEM imaging medium (e.g., Life Technologies) This live-cell visualization medium is supplemented with 2 mM glutamine, 1% penicillin–streptomycin, and 10% FBS.
Streptavidin-coated quantum dots (QD-SAVs) (Life Technologies) Equipment
Attofluor cell chamber (Life Technologies A-7816) Autoclave Coverslips (circular; 18-mm diameter; sterile) Culture dish Forceps (sterile) Parafilm Plastic box Staining jar (or mini-rack) Thermobloc Tissues (sterile) Tubes (sterile; 15 mL and 50 mL; Falcon) Vortex mixer Water baths (preset to 37˚C and 80˚C) METHOD Preparation of Hypertonic Loading Medium Containing QD-Ps
1. Prewarm 5 mL of DMEM medium, without serum, to 37˚C.
2. Melt the polyethylene glycol (waxy solid on top of sucrose crystals) contained in the tube of Influx Pinocytic Cell-Loading Reagent by heating the tube in hot water (80˚C) for 2 min. 3. Remove the cap of the tube and quickly add 4.7 mL of DMEM medium, without serum, at 37˚C.
4. Replace the cap and vortex the tube vigorously to completely dissolve the sucrose crystals.
5. Once the medium is homogeneous, add 250 µL of FBS and 50 µL of 1 M HEPES buffer at pH 7.4 (or other suitable buffer, depending on the cell type). Replace the cap and vortex the tube for a few minutes. The hypertonic medium can be stored at 4˚C for up to several weeks. Keep the tube capped until immediately before use. Just before the experiments, open the tube in a sterile environment (e.g., close to a flame) to keep the hypertonic medium sterile. Check the color of the medium, which should be red if no contamination is found, assuming that the pH indicator is phenol red.
6. Immediately before use, prepare the QD-Ps (coupled reaction product) by incubating QD-SAVs (at a final concentration of 1 nM) with biotinylated protein (ideally in a 1:1 molar ratio) in the hypertonic medium. Internalization of QD-Ps into Live Cultured Cells The cell-loading technique used here is based on the procedure described by Okada and Rechsteiner (1982).
7. Plate cultured cells at a density of 1.5 × 105 cells/cm2 on 18-mm glass coverslips that have been maintained in DMEM medium containing 10% FBS at 37˚C in a 5% CO2 atmosphere.
8. Prewarm 100 µL each of hypertonic medium containing QD-Ps, hypotonic lysis medium, and MEM imaging medium to 37˚C (assuming that this is the ideal temperature for the cell type). 9. Use sterile forceps to collect a coverslip from the culture dish.
10. Remove the excess medium by touching the edge of the coverslip to a sterile tissue. Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078238
1077
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on December 15, 2014 - Published by Cold Spring Harbor Laboratory Press
S. Courty and M. Dahan
FIGURE 1. Detection of single intracellular QD-tagged proteins. (A) Bright-field image of a cultured HeLa cell. (B) Fluorescence image of individual QDs internalized in the cytoplasm. (C,D) In the case of small fluorescent dextran molecules internalized with the same protocol, the molecules diffuse rapidly in the cytoplasm, leading to a uniform staining.
11. Place the coverslip cell-side up on a piece of parafilm (5 × 5 cm) heated at 37˚C with a thermobloc. 12. Quickly but gently deposit 100 µL of hypertonic medium containing QD-Ps on the coverslip boundary. 13. Incubate the cells for 10 min at 37˚C in the hypertonic medium along with QD-Ps. Cover the coverslip with a plastic box to avoid evaporation. See Troubleshooting.
14. Repeat Step 10. 15. Place the coverslip vertically in a staining jar (alternatively, a mini-rack can be used), filled with at least 7 mL of prewarmed hypotonic lysis medium, for exactly 2 min. After osmotic lysis of pinocytic vesicles, induced by incubating the cells in a hypotonic lysis medium, QD-Ps are released homogeneously in the cell cytosol. See Troubleshooting.
16. Repeat Step 10. 17. Place the coverslip in the cell chamber filled with MEM imaging medium prewarmed to 37˚C.
18. Incubate the cells for 10 min at 37˚C to promote recovery.
19. Image the cells, using appropriate settings.
Figure 1 shows an example of detection of single intracellular QD-tagged proteins. See Troubleshooting.
TROUBLESHOOTING Problem (Step 13): Cells do not adhere to coverslips. Solution: For nonadhesive cells (e.g., Drosophila cells), use the following procedure for coating
coverslips with poly-L-lysine (this may be important for Step 15 in particular because the viscosity of the hypertonic medium may remove cells from the glass coverslip): 1. Autoclave the coverslips to be used. 2. Rinse the coverslips with phosphate-buffered saline (PBS) to remove any debris and to uniformly wet their surfaces. 3. Cover the surface of the coverslip with 200 µL of a solution of poly-L-lysine (1 mg/mL; SigmaAldrich). 1078
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078238
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on December 15, 2014 - Published by Cold Spring Harbor Laboratory Press
Tracking Intracellular Proteins Using QDs
4. Incubate the coverslips in poly-L-lysine for at least 1 h. 5. Aspirate and allow the coverslips to dry completely (overnight if necessary). 6. Gently wash the coverslips (at least five times) with PBS to remove any excess poly-L-lysine, which appears to be very toxic to cells. 7. Store poly-L-lysine-coated coverslips in PBS at 4˚C for up to 2 wk in a clean, dry place to avoid dust and humidity until needed. Problem (Step 15): Cell viability is low. Solution: Reduce the exposure time in the hypotonic lysis medium; too long an exposure may result in
blebbing of the cell membrane. Problem (Step 19): There is cell autofluorescence. Solution: Selecting the right QD emission wavelength is important for overcoming cell autofluor-
escence. Cells show autofluorescence composed of at least four distinct excitation and emission maxima from endogenous metabolites: the tryptophan peak (290-nm excitation, 330-nm emission), the NAD(P)H peak (350-nm excitation, 450-nm emission), the riboflavin (FAD) peak (450-nm excitation, 530-nm emission), and a yet unidentified peak (500-nm excitation, 530-nm emission) (Heintzelman et al. 2000). Note also that autofluorescence varies greatly in living cells. For example, autofluorescence is low in freshly prepared cells and increases with time during culture until a plateau is reached. To avoid any overlap with the cell autofluorescence, we thus recommend using QDs emitting in the red range at 655 nm. The main difficulty in tracking single QDs inside living cells comes from their three-dimensional motion, which might lead to defocusing of the fluorescence spots. Unfortunately, no general method currently exists to overcome this problem. We thus recommend, whenever possible, to focus on a part where the cells are relatively flat on the coverslip. RELATED INFORMATION
This protocol has been successfully implemented for tracking single biotinylated-kinesin molecules coupled to QD-SAVs and internalized within living cultured cells (Courty et al. 2006). Single-particle tracking has also proven to be a powerful method for deciphering the molecular organization of the plasma membrane (see Tracking Individual Membrane Proteins Using Quantum Dots [Courty and Dahan 2013a]). For a discussion of practical issues related to the detection of individual nanoparticles by wide-field fluorescence microscopy, see Ultrasensitive Imaging in Live Cells Using Fluorescent Quantum Dots (Courty and Dahan 2013b). REFERENCES Courty S, Luccardini C, Bellaiche Y, Cappello G, Dahan M. 2006. Tracking individual kinesin motors in living cells using single quantum-dot imaging. Nano Lett 6: 1491–1495. Courty S, Dahan M. 2013a. Tracking individual membrane proteins using quantum dots. Cold Spring Harb Protoc doi: 10.1101/pdb.prot078196. Courty S, Dahan M. 2013b. Ultrasensitive imaging in live cells using fluorescent quantum dots. Cold Spring Harb Protoc doi: 10.1101/pdb. top078220.
Heintzelman DL, Lotan R, Richards-Kortum RR. 2000. Characterization of the autofluorescence of polymorphonuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia. Photochem Photobiol 71: 327–332. Okada CY, Rechsteiner M. 1982. Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell 29: 33–41.
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078238
1079
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CALIF-SF on December 15, 2014 - Published by Cold Spring Harbor Laboratory Press
Tracking Individual Intracellular Proteins Using Quantum Dots Sébastien Courty and Maxime Dahan Cold Spring Harb Protoc; doi: 10.1101/pdb.prot078238 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Cell Biology, general (1069 articles) Cell Imaging (464 articles) Fluorescence (425 articles) Fluorescence, general (287 articles) In Vivo Imaging (280 articles) Live Cell Imaging (243 articles) Tagging Proteins (51 articles) Video Imaging / Time Lapse Imaging (164 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2013 Cold Spring Harbor Laboratory Press
