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Protocol
Detection of Bak/Bax Activating Conformation Change by Intracellular Flow Cytometry Grant Dewson1 Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia
Like the commonly used immunoprecipitation (IP) approach, this procedure for the detection of activated Bak or Bax by intracellular flow cytometry is based on the principle that Bak and Bax, during activation, expose occluded amino-terminal epitopes that can be recognized by conformation-specific antibodies. Flow cytometric analysis requires fewer cells and is less time-consuming than IP. Further, in contrast to IP, flow cytometry produces a quantifiable assessment of the percentage of cells containing activated Bak or Bax, which can be correlated with cell death.
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 materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Antibodies Primary antibody, conformation-specific (for Bak and/or Bax; see Table 1) Secondary antibody, fluorochrome-conjugated (e.g., FITC or RPE conjugate; Molecular Probes) Apoptotic stimulus Cell line of interest (e.g., mouse embryonic fibroblasts [MEF]), pretreated with a broad-range caspase inhibitor We routinely perform this procedure using 1 × 10 5 MEF. In cells that will be treated with a death stimulus, caspases should first be blocked with a broad-range caspase inhibitor, such as qVD-OPh or zVAD-fmk (Enzyme Systems).
Intracellular FACS buffer Paraformaldehyde (PFA) Prepare a 4% solution in 1× phosphate-buffered saline (PBS). Dissolve by heating at 60˚C and add 1 M NaOH dropwise until solution clears. Dilute to 1% PFA in 1× PBS immediately before use (Step 4).
Phosphate-buffered saline (PBS) (1×; Ca2+- and Mg2+-free) (ice-cold) 1
Correspondence: [email protected]
© 2015 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086462
477
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on May 11, 2015 - Published by Cold Spring Harbor Laboratory Press
G. Dewson
TABLE 1. Commonly used conformation-specific Bak or Bax antibodies Antibody (species reactivity)
Species
Source
Epitope/ immunogen
Bax
6A7 (h, m, r) NT (h, m) N20 (h, m, r) Clone 3 (h)
Mouse Rabbit Rabbit Mouse
Santa Cruz Biotech BD Biosciences Santa Cruz Biotech BD Biosciences
aa12–24 aa1–21 Amino terminus aa55–178
Bak
Ab-1 (h) NT (h, m, r) 22–38 (h, m)
Mouse Rabbit Rabbit
Millipore Millipore Sigma-Aldrich
Amino terminus aa23–38 aa23–38
References Hsu and Youle 1998; Nechushtan et al. 1999 Vogel et al. 2012 Khaled et al. 1999 Dewson et al. 2003 Griffiths et al. 1999 Oberle et al. 2010
h, human; m, mouse; r, rat.
Equipment
Centrifuge (with fixed-angle and swing-out rotors) at 4˚C Centrifuge tubes FACs analyzer Flow cytometry tubes Rotating wheel at 4˚C
METHOD
1. Treat cells (1 × 105) with an apoptotic stimulus as required. Beginning the procedure with excess cells (1 × 10 5) eases sample processing and improves FACS profiles.
2. Harvest the cells by centrifugation at 2500g for 5 min. Discard the supernatant. 3. Wash the cell pellet once in ice-cold 1× PBS. Centrifuge at 2500g for 5 min and discard the supernatant. 4. Resuspend the cell pellet in 1% PFA for 15 min at room temperature to fix cells. 5. Repeat Steps 2 and 3. Once cells are fixed, subsequent centrifugation steps are best performed in a swing-out (rather than fixedangle) rotor to ensure the cells pellet at the bottom of the tube.
6. Resuspend the cell pellet in 100 µL of primary antibody at 1 µg/mL in intracellular FACS buffer. Include a “no primary antibody” control for all samples. Incubate the samples on a rotating wheel for 1 h at 4˚C.
7. Add 1 mL of intracellular FACS buffer to each sample.
8. Centrifuge at 2500g for 5 min and discard the supernatant. 9. Resuspend each sample in 100 µL of species-specific FITC- (or RPE)-conjugated secondary antibody at 1 µg/mL in intracellular FACS buffer. Incubate on a rotating wheel for 1 h at 4˚C in the dark. 10. Repeat Steps 7 and 8. 11. Resuspend each sample in 200 µL of ice-cold 1× PBS. Transfer to flow cytometry tubes and process using the FACS analyzer. See Troubleshooting.
TROUBLESHOOTING Problem (Step 11): The detectable shift in fluorescence is limited. 478
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086462
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on May 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Detecting Bak/Bax Activation by FACS
Solution: The shift in fluorescence depends on Bak/Bax expression levels and the efficiency of acti-
vation. Detection can be improved using a biotinylated secondary antibody followed by signal amplification with streptavidin-FITC/RPE. Problem (Step 11): The entire cell population shifts during apoptosis. Solution: Given the stochastic nature of a cell death response, one should expect to detect a population
of cells with activated Bak or Bax that is dependent both on the time and dose of apoptotic stimulus. If the entire population shifts due to treatment, consider the following.
• All cells have activated Bak/Bax. Based on a death assay, for example, propidium iodide uptake, perform apoptosis induction using submaximal conditions.
• There is nonspecific uptake of the secondary antibody in treated cells. Be sure to include a “no primary antibody” control for all samples and trial different conformation-specific antibodies. Problem (Step 11): The percentage of cells with activated Bak/Bax is lower than the percentage of
“dead” cells.
Solution: As Bak/Bax activation is a relatively early event during apoptosis, it should precede cell death
as monitored by propidium iodide uptake or AnnexinV positivity. If this is not the case, it may indicate the death stimulus used is not Bak/Bax-dependent. Alternatively, the concentration and time of death induction can be reduced and cells pretreated with caspase inhibitors to ensure that detection of activated Bak/Bax is not obscured by late-stage caspase-mediated destruction. RELATED TECHNIQUES
Flow cytometric analysis of Bak conformation change can be performed in conjunction with flow cytometric analysis of cytochrome c release (Waterhouse et al. 2004). The latter protocol involves permeabilization of the cells with digitonin before fixation and is amenable to assessing conformation change of Bak but not Bax, as the cytosolic population of Bax is lost before fixation. RECIPES Intracellular FACS Buffer
1× phosphate-buffered saline (Ca2+- and Mg2+-free) 0.5% bovine serum albumin 0.1% saponin Phosphate-Buffered Saline (PBS)
Amount to add (for 1× solution)
Final concentration (1×)
Amount to add (for 10× stock)
Final concentration (10×)
8g 0.2 g 1.44 g 0.24 g
137 mM 2.7 mM 10 mM 1.8 mM
80 g 2g 14.4 g 2.4 g
1.37 M 27 mM 100 mM 18 mM
If necessary, PBS may be supplemented with the following: CaCl2•2H2O 0.133 g 1 mM 1.33 g MgCl2•6H2O 0.10 g 0.5 mM 1.0 g
10 mM 5 mM
Reagent NaCl KCl Na2HPO4 KH2PO4
PBS can be made as a 1× solution or as a 10× stock. To prepare 1 L of either 1× or 10× PBS, dissolve the reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086462
479
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on May 11, 2015 - Published by Cold Spring Harbor Laboratory Press
G. Dewson
ACKNOWLEDGMENTS
G.D. is supported by the National Health and Medical Research Council of Australia (637335), Australian Research Council (FT100100791), and the Association for International Cancer Research (10-230). The present work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.
REFERENCES Dewson G, Snowden RT, Almond JB, Dyer MJ, Cohen GM. 2003. Conformational change and mitochondrial translocation of Bax accompany proteasome inhibitor-induced apoptosis of chronic lymphocytic leukemic cells. Oncogene 22: 2643–2654. Griffiths GJ, Dubrez L, Morgan CP, Jones NA, Whitehouse J, Corfe BM, Dive C, Hickman JA. 1999. Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol 144: 903–914. Hsu Y-T, Youle RJ. 1998. Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J Biol Chem 273: 10777–10783. Khaled AR, Kim K, Hofmeister R, Muegge K, Durum SK. 1999. Withdrawal of IL-7 induces bax translocation from cytosol to mitochondria through a rise in intracellular pH. Proc Natl Acad Sci 96: 14476–14481.
480
Nechushtan A, Smith CL, Hsu YT, Youle RJ. 1999. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J 18: 2330–2341. Oberle C, Huai J, Reinheckel T, Tacke M, Rassner M, Ekert PG, Buellesbach J, Borner C. 2010. Lysosomal membrane permeabilization and cathepsin release is a Bax/Bak-dependent, amplifying event of apoptosis in fibroblasts and monocytes. Cell Death Differ 17: 1167– 1178. Vogel S, Raulf N, Bregenhorn S, Biniossek ML, Maurer U, Czabotar P, Borner C. 2012. Cytosolic Bax: Does it require binding proteins to keep its pro-apoptotic activity in check? J Biol Chem 287: 9112– 9127. Waterhouse NJ, Steel R, Kluck R, Trapani JA. 2004. Assaying cytochrome c translocation during apoptosis. Methods Mol Biol 284: 307–313.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086462
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on May 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Detection of Bak/Bax Activating Conformation Change by Intracellular Flow Cytometry Grant Dewson Cold Spring Harb Protoc; doi: 10.1101/pdb.prot086462 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 (1108 articles) Characterization of Protein Complexes (70 articles) Characterization of Proteins (176 articles) Flow Cytometry (20 articles) Protein Expression and Interactions (65 articles)
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© 2015 Cold Spring Harbor Laboratory Press
Protocol
Detection of Bak/Bax Activating Conformation Change by Intracellular Flow Cytometry Grant Dewson1 Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia
Like the commonly used immunoprecipitation (IP) approach, this procedure for the detection of activated Bak or Bax by intracellular flow cytometry is based on the principle that Bak and Bax, during activation, expose occluded amino-terminal epitopes that can be recognized by conformation-specific antibodies. Flow cytometric analysis requires fewer cells and is less time-consuming than IP. Further, in contrast to IP, flow cytometry produces a quantifiable assessment of the percentage of cells containing activated Bak or Bax, which can be correlated with cell death.
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 materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Antibodies Primary antibody, conformation-specific (for Bak and/or Bax; see Table 1) Secondary antibody, fluorochrome-conjugated (e.g., FITC or RPE conjugate; Molecular Probes) Apoptotic stimulus Cell line of interest (e.g., mouse embryonic fibroblasts [MEF]), pretreated with a broad-range caspase inhibitor We routinely perform this procedure using 1 × 10 5 MEF. In cells that will be treated with a death stimulus, caspases should first be blocked with a broad-range caspase inhibitor, such as qVD-OPh or zVAD-fmk (Enzyme Systems).
Intracellular FACS buffer Paraformaldehyde (PFA) Prepare a 4% solution in 1× phosphate-buffered saline (PBS). Dissolve by heating at 60˚C and add 1 M NaOH dropwise until solution clears. Dilute to 1% PFA in 1× PBS immediately before use (Step 4).
Phosphate-buffered saline (PBS) (1×; Ca2+- and Mg2+-free) (ice-cold) 1
Correspondence: [email protected]
© 2015 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086462
477
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on May 11, 2015 - Published by Cold Spring Harbor Laboratory Press
G. Dewson
TABLE 1. Commonly used conformation-specific Bak or Bax antibodies Antibody (species reactivity)
Species
Source
Epitope/ immunogen
Bax
6A7 (h, m, r) NT (h, m) N20 (h, m, r) Clone 3 (h)
Mouse Rabbit Rabbit Mouse
Santa Cruz Biotech BD Biosciences Santa Cruz Biotech BD Biosciences
aa12–24 aa1–21 Amino terminus aa55–178
Bak
Ab-1 (h) NT (h, m, r) 22–38 (h, m)
Mouse Rabbit Rabbit
Millipore Millipore Sigma-Aldrich
Amino terminus aa23–38 aa23–38
References Hsu and Youle 1998; Nechushtan et al. 1999 Vogel et al. 2012 Khaled et al. 1999 Dewson et al. 2003 Griffiths et al. 1999 Oberle et al. 2010
h, human; m, mouse; r, rat.
Equipment
Centrifuge (with fixed-angle and swing-out rotors) at 4˚C Centrifuge tubes FACs analyzer Flow cytometry tubes Rotating wheel at 4˚C
METHOD
1. Treat cells (1 × 105) with an apoptotic stimulus as required. Beginning the procedure with excess cells (1 × 10 5) eases sample processing and improves FACS profiles.
2. Harvest the cells by centrifugation at 2500g for 5 min. Discard the supernatant. 3. Wash the cell pellet once in ice-cold 1× PBS. Centrifuge at 2500g for 5 min and discard the supernatant. 4. Resuspend the cell pellet in 1% PFA for 15 min at room temperature to fix cells. 5. Repeat Steps 2 and 3. Once cells are fixed, subsequent centrifugation steps are best performed in a swing-out (rather than fixedangle) rotor to ensure the cells pellet at the bottom of the tube.
6. Resuspend the cell pellet in 100 µL of primary antibody at 1 µg/mL in intracellular FACS buffer. Include a “no primary antibody” control for all samples. Incubate the samples on a rotating wheel for 1 h at 4˚C.
7. Add 1 mL of intracellular FACS buffer to each sample.
8. Centrifuge at 2500g for 5 min and discard the supernatant. 9. Resuspend each sample in 100 µL of species-specific FITC- (or RPE)-conjugated secondary antibody at 1 µg/mL in intracellular FACS buffer. Incubate on a rotating wheel for 1 h at 4˚C in the dark. 10. Repeat Steps 7 and 8. 11. Resuspend each sample in 200 µL of ice-cold 1× PBS. Transfer to flow cytometry tubes and process using the FACS analyzer. See Troubleshooting.
TROUBLESHOOTING Problem (Step 11): The detectable shift in fluorescence is limited. 478
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086462
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on May 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Detecting Bak/Bax Activation by FACS
Solution: The shift in fluorescence depends on Bak/Bax expression levels and the efficiency of acti-
vation. Detection can be improved using a biotinylated secondary antibody followed by signal amplification with streptavidin-FITC/RPE. Problem (Step 11): The entire cell population shifts during apoptosis. Solution: Given the stochastic nature of a cell death response, one should expect to detect a population
of cells with activated Bak or Bax that is dependent both on the time and dose of apoptotic stimulus. If the entire population shifts due to treatment, consider the following.
• All cells have activated Bak/Bax. Based on a death assay, for example, propidium iodide uptake, perform apoptosis induction using submaximal conditions.
• There is nonspecific uptake of the secondary antibody in treated cells. Be sure to include a “no primary antibody” control for all samples and trial different conformation-specific antibodies. Problem (Step 11): The percentage of cells with activated Bak/Bax is lower than the percentage of
“dead” cells.
Solution: As Bak/Bax activation is a relatively early event during apoptosis, it should precede cell death
as monitored by propidium iodide uptake or AnnexinV positivity. If this is not the case, it may indicate the death stimulus used is not Bak/Bax-dependent. Alternatively, the concentration and time of death induction can be reduced and cells pretreated with caspase inhibitors to ensure that detection of activated Bak/Bax is not obscured by late-stage caspase-mediated destruction. RELATED TECHNIQUES
Flow cytometric analysis of Bak conformation change can be performed in conjunction with flow cytometric analysis of cytochrome c release (Waterhouse et al. 2004). The latter protocol involves permeabilization of the cells with digitonin before fixation and is amenable to assessing conformation change of Bak but not Bax, as the cytosolic population of Bax is lost before fixation. RECIPES Intracellular FACS Buffer
1× phosphate-buffered saline (Ca2+- and Mg2+-free) 0.5% bovine serum albumin 0.1% saponin Phosphate-Buffered Saline (PBS)
Amount to add (for 1× solution)
Final concentration (1×)
Amount to add (for 10× stock)
Final concentration (10×)
8g 0.2 g 1.44 g 0.24 g
137 mM 2.7 mM 10 mM 1.8 mM
80 g 2g 14.4 g 2.4 g
1.37 M 27 mM 100 mM 18 mM
If necessary, PBS may be supplemented with the following: CaCl2•2H2O 0.133 g 1 mM 1.33 g MgCl2•6H2O 0.10 g 0.5 mM 1.0 g
10 mM 5 mM
Reagent NaCl KCl Na2HPO4 KH2PO4
PBS can be made as a 1× solution or as a 10× stock. To prepare 1 L of either 1× or 10× PBS, dissolve the reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086462
479
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on May 11, 2015 - Published by Cold Spring Harbor Laboratory Press
G. Dewson
ACKNOWLEDGMENTS
G.D. is supported by the National Health and Medical Research Council of Australia (637335), Australian Research Council (FT100100791), and the Association for International Cancer Research (10-230). The present work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.
REFERENCES Dewson G, Snowden RT, Almond JB, Dyer MJ, Cohen GM. 2003. Conformational change and mitochondrial translocation of Bax accompany proteasome inhibitor-induced apoptosis of chronic lymphocytic leukemic cells. Oncogene 22: 2643–2654. Griffiths GJ, Dubrez L, Morgan CP, Jones NA, Whitehouse J, Corfe BM, Dive C, Hickman JA. 1999. Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol 144: 903–914. Hsu Y-T, Youle RJ. 1998. Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J Biol Chem 273: 10777–10783. Khaled AR, Kim K, Hofmeister R, Muegge K, Durum SK. 1999. Withdrawal of IL-7 induces bax translocation from cytosol to mitochondria through a rise in intracellular pH. Proc Natl Acad Sci 96: 14476–14481.
480
Nechushtan A, Smith CL, Hsu YT, Youle RJ. 1999. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J 18: 2330–2341. Oberle C, Huai J, Reinheckel T, Tacke M, Rassner M, Ekert PG, Buellesbach J, Borner C. 2010. Lysosomal membrane permeabilization and cathepsin release is a Bax/Bak-dependent, amplifying event of apoptosis in fibroblasts and monocytes. Cell Death Differ 17: 1167– 1178. Vogel S, Raulf N, Bregenhorn S, Biniossek ML, Maurer U, Czabotar P, Borner C. 2012. Cytosolic Bax: Does it require binding proteins to keep its pro-apoptotic activity in check? J Biol Chem 287: 9112– 9127. Waterhouse NJ, Steel R, Kluck R, Trapani JA. 2004. Assaying cytochrome c translocation during apoptosis. Methods Mol Biol 284: 307–313.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086462
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on May 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Detection of Bak/Bax Activating Conformation Change by Intracellular Flow Cytometry Grant Dewson Cold Spring Harb Protoc; doi: 10.1101/pdb.prot086462 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 (1108 articles) Characterization of Protein Complexes (70 articles) Characterization of Proteins (176 articles) Flow Cytometry (20 articles) Protein Expression and Interactions (65 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2015 Cold Spring Harbor Laboratory Press
