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Protocol
Measurement of Intracellular Ca2+ Release in Intact Cells Using 45Ca2+ Tomas Luyten, Geert Bultynck, Jan B. Parys, Humbert De Smedt, and Ludwig Missiaen1 Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, KU Leuven Campus Gasthuisberg O&N I, 3000 Leuven, Belgium
This protocol describes a technique using 45Ca2+ to measure the release of Ca2+ from the intracellular stores in monolayers of intact cells cultured in 12-well 4-cm2 clusters. The 45Ca2+-flux technique described here can only be applied to cell types that adhere to plastic. We describe the loading of the stores with 45Ca2+, and the subsequent 45Ca2+ efflux.
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
Ca2+-containing modified Krebs solution Cell line of interest and appropriate culture medium (see Step 1) Efflux medium for intact cells Prepare efflux medium with appropriate additions (e.g., an extracellular agonist coupled to IP3 production, thapsigargin, or A23187). See Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca2+ (Missiaen et al. 2014).
Loading medium for intact cells Scintillation liquid (H2O-compatible) Sodium dodecyl sulfate (SDS; 2% w/v)
Equipment
Cell-culture plate (12-well) Mechanical shaker Scintillation counter and vials Thermostatic bath with circulator to a plate and holders for solutions 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
284
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on June 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Measuring Ca 2+ Release in Intact Cells
Cell-culture incubator Vacuum pump
METHOD
1. Plate desired cell line in wells of a 12-well cell-culture plate and grow cells for the appropriate length of time in a cell-culture incubator. We normally plate the cells at a density of 15,000 (MEF cells), 30,000 (COS-1 cells), or 60,000 (HeLa cells) cells per well and let them grow for 7 d. A7r5 cells plated at a density of 40,000 cells per well are grown for 10 d.
2. Before starting the 45Ca2+ flux, set the temperature of the thermostatic bath at 25˚C, and start the circulation to the thermostatic plate and the holders of the various solutions. Ca2+ fluxes in intact cells can also be performed at 37˚C.
45
3. Place the Ca2+-containing modified Krebs solution, the loading medium, the efflux medium (with or without additions), and the SDS in their appropriate holders at 25˚C.
4. Fill racks with scintillation vials and add 7 mL of scintillation liquid at room temperature to each vial. We perform a 45Ca2+-efflux experiment in each of the 12 wells of cell-culture plate. We add and remove efflux medium 10 times and finally collect the remaining radioactivity. Therefore, 132 scintillation vials are needed.
5. Transfer the 12-well plate with the cells in culture medium from an incubator to the thermostatic plate at 25˚C on a mechanical shaker. 6. Remove the culture medium with a pipette tip connected to a vacuum pump.
The use of a vacuum pump to aspirate the medium instead of a pipette increases the speed of the procedure. See Troubleshooting.
7. Wash each well once with 1 mL of Ca2+-containing modified Krebs solution at 25˚C (i.e., add this medium on top of the cells and immediately remove it with a pipette tip connected to a vacuum pump). See Troubleshooting.
8. Add 1 mL of loading medium to each well at 25˚C to load the stores in the cells with 45Ca2+, and incubate for sufficient time to obtain a steady-state loading. Gently shake the cells. Users should familiarize themselves with how to handle radioactive chemicals. Steady-state loading is usually reached after 1 h, but should be determined empirically. See Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca2+ (Missiaen et al. 2014).
9. Remove the loading medium with a pipette tip connected to a vacuum pump. 10. Wash each well once or twice with 1 mL of efflux medium at 25˚C.
11. Add 0.5-1 mL of efflux medium to each well at 25˚C. Gently shake the cells.
12. After 2 min, remove the efflux medium with a pipette and transfer it to a scintillation vial. See Troubleshooting.
13. Repeat Steps 11 and 12. Because the efflux medium is removed and replaced by new medium each time, it is possible to change its composition (e.g., by adding and subsequently removing an extracellular agonist coupled to IP3 production). The duration of the efflux can be variable, but we normally take samples for 20 min. Use a different pipette for removing the efflux medium and for subsequently adding new efflux medium.
14. At the end of the efflux, add 1 mL of a solution with SDS at 25˚C for 30 min to solubilize all 45 Ca2+ remaining in the stores. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
285
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T. Luyten et al.
15. Remove the solution with a pipette tip and transfer it to a scintillation vial. 16. Count the radioactivity in a scintillation counter. TROUBLESHOOTING Problem (Step 6): Adherent cells are sucked up during the removal of medium. Solution: Never touch the cells on the bottom of the well with the pipette tip. We remove the medium
by tilting the 12-well plate during medium removal and by placing the pipette tip against the lateral wall about 2 mm above the bottom. See Figure 1 in Measurement of Intracellular Ca2+ Release in Permeabilized Cells Using 45Ca2+ (Luyten et al. 2014). Problem (Step 7): The cells detach. Solution: This protocol can only be applied to cells that adhere well to the 12-well cell-culture plates.
Some cell types detach during the various washing steps. One must therefore check whether the cells remain fixed during the whole procedure. One can detect the loss of cells by visual inspection of the remaining monolayer during the flux, by phase-contrast microscopy at the end of the efflux before adding the SDS, or by a sudden noninduced increase in the rate of 45Ca2+ efflux caused by the detachment of radioactive cells. Visual inspection is only possible for some cells like COS-1 cells. Other cells like HeLa cells are less visible and still other cells like ciPTEC cells (conditionally immortalized proximal-tubule epithelial cells) are not visible at all by the naked eye. It sometimes helps one to plate the cells in wells coated with e.g., collagen, poly-L-lysine or gelatin. Problem (Step 12): There is splashing of radioactivity outside the scintillation vial, or the vials turn
sideways in the rack, losing their contents. Solution: Both problems can be avoided by filling the vials with scintillation fluid before starting
the efflux. DISCUSSION
This 45Ca2+-efflux experiment (with the exception of the counting of the radioactivity) takes maximally 2 h. Basic procedures of cell culturing and working with radioactivity are not covered and it is assumed that the reader is familiar with them. For a detailed discussion of this technique see Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca 2+ (Missiaen et al. 2014). The efflux experiment is performed in the absence of extracellular Ca2+ to exclude the contribution of the influx of Ca2+ across the plasma membrane. The agonist-induced increase in the rate of 45 Ca2+ extrusion depends on the activation of the PMCA Ca2+ pumps and of the Na+–Ca2+ exchangers by the increased [Ca2+]cyt resulting from the various Ca2+ fluxes in and out of the various stores. Therefore, a limitation of the technique is that the signal is only a very indirect measure of the intracellular Ca2+ release. It is furthermore impossible in intact cells to eliminate the contribution of some organelles like the mitochondria, because they are needed to produce the ATP that fuels the Ca2+ pumps. The major advantage of experiments in intact cells is that the IP3R can be studied in its natural environment in the presence of all known or unknown regulators of the channel and with feedback of released Ca2+ on its own release. This technique is described for the sake of being comprehensive, but is not in frequent current use. It can be useful in studies of intracellular Ca2+ release where the use of cytosolic Ca2+ dyes is less obvious, for example, when a Ca2+-releasing agent like caffeine interferes with the fluorescence (Van Acker et al. 2004) or when changes in extracellular tonicity occur (Missiaen et al. 1996). Cytosolic Ca2+ dyes are less useful in the latter condition because osmotic fluxes of H2O across the plasma membrane change the dye concentration and the ionic composition in the cell and therefore interfere with the emitted fluorescence. 286
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on June 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Measuring Ca 2+ Release in Intact Cells
RECIPES Ca2+-Containing Modified Krebs Solution
Reagent
Final concentration
NaCl KCl MgCl2 HEPES Glucose CaCl2
135 mM 5.9 mM 1.2 mM 11.6 mM 11.5 mM 1.5 mM
Dissolve chemicals in distilled water and adjust the pH to 7.3 with 10 M NaOH. Store at 4˚C for 1 mo maximum. Efflux Medium for Intact Cells
Reagent
Final concentration
NaCl KCl MgCl2 HEPES Glucose EGTA a A23187 (2 mM in ethanol) a Thapsigargin (2 mM in ethanol)
135 mM 5.9 mM 1.2 mM 11.6 mM 11.5 mM 2 mM 10 µM 2 µM
Dissolve all chemicals in distilled water and adjust the pH to 7.3 with 10 M NaOH. Store at 4˚C for 1 mo maximum. a
Optional agents that can be added to the standard efflux medium.
Loading Medium for Intact Cells
Reagent NaCl KCl MgCl2 HEPES Glucose 40 CaCl2 + 45CaCl2
Final concentration 135 mM 5.9 mM 1.2 mM 11.6 mM 11.5 mM 0.2 mM
Dissolve chemicals in distilled water and adjust the pH to 7.3 with 10 M NaOH. Store medium (without 45CaCl2) at 4˚C for 1 mo maximum.
The 45Ca2+ should have a specific activity of 0.5 MBq/mL. For example, use 14 µM 45Ca2+ from PerkinElmer (NEZ013; specific activity of 802.65 MBq/mg; concentration of 1465.77 MBq/mL) and 186 µM 40Ca2+.
ACKNOWLEDGMENTS
We thank Marina Crabbe, Anja Florizoone, and Kirsten Welkenhuyzen for excellent technical help. This work was supported by the Research Council of the KU Leuven via the Concerted Actions Program (GOA/09/012) and by the Interuniversity Attraction Poles Program (Belgian Science Policy; P6/28). Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
287
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on June 11, 2015 - Published by Cold Spring Harbor Laboratory Press
T. Luyten et al.
REFERENCES Luyten T, Bultynck G, Parys JB, De Smedt H, Missiaen L. 2014. Measurement of intracellular Ca2+ release in permeabilized cells using 45Ca2+. Cold Spring Harb Protoc doi: 10.1101/pdb.prot073189. Missiaen L, De Smedt H, Parys JB, Sienaert I, Vanlingen S, Droogmans G, Nilius B, Casteels R. 1996. Hypotonically induced calcium release from intracellular calcium stores. J Biol Chem 271: 4601–4604. Missiaen L, Luyten T, Bultynck G, Parys JB, De Smedt H. 2014. Measurement of intracellular Ca2+ release in intact and permeabi-
288
lized cells using 45Ca2+. Cold Spring Harb Protoc doi: 10.1101/pdb. top066126. Van Acker K, Bultynck G, Rossi D, Sorrentino V, Boens N, Missiaen L, De Smedt H, Parys JB, Callewaert G. 2004. The 12 kDa FK506-binding protein, FKBP12, modulates the Ca2+-flux properties of the type-3 ryanodine receptor. J Cell Sci 117: 1129–1137.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on June 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Measurement of Intracellular Ca2+ Release in Intact Cells Using 45Ca2+ Tomas Luyten, Geert Bultynck, Jan B. Parys, Humbert De Smedt and Ludwig Missiaen Cold Spring Harb Protoc; doi: 10.1101/pdb.prot073197 Email Alerting Service Subject Categories
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© 2014 Cold Spring Harbor Laboratory Press
Protocol
Measurement of Intracellular Ca2+ Release in Intact Cells Using 45Ca2+ Tomas Luyten, Geert Bultynck, Jan B. Parys, Humbert De Smedt, and Ludwig Missiaen1 Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, KU Leuven Campus Gasthuisberg O&N I, 3000 Leuven, Belgium
This protocol describes a technique using 45Ca2+ to measure the release of Ca2+ from the intracellular stores in monolayers of intact cells cultured in 12-well 4-cm2 clusters. The 45Ca2+-flux technique described here can only be applied to cell types that adhere to plastic. We describe the loading of the stores with 45Ca2+, and the subsequent 45Ca2+ efflux.
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
Ca2+-containing modified Krebs solution Cell line of interest and appropriate culture medium (see Step 1) Efflux medium for intact cells Prepare efflux medium with appropriate additions (e.g., an extracellular agonist coupled to IP3 production, thapsigargin, or A23187). See Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca2+ (Missiaen et al. 2014).
Loading medium for intact cells Scintillation liquid (H2O-compatible) Sodium dodecyl sulfate (SDS; 2% w/v)
Equipment
Cell-culture plate (12-well) Mechanical shaker Scintillation counter and vials Thermostatic bath with circulator to a plate and holders for solutions 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
284
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on June 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Measuring Ca 2+ Release in Intact Cells
Cell-culture incubator Vacuum pump
METHOD
1. Plate desired cell line in wells of a 12-well cell-culture plate and grow cells for the appropriate length of time in a cell-culture incubator. We normally plate the cells at a density of 15,000 (MEF cells), 30,000 (COS-1 cells), or 60,000 (HeLa cells) cells per well and let them grow for 7 d. A7r5 cells plated at a density of 40,000 cells per well are grown for 10 d.
2. Before starting the 45Ca2+ flux, set the temperature of the thermostatic bath at 25˚C, and start the circulation to the thermostatic plate and the holders of the various solutions. Ca2+ fluxes in intact cells can also be performed at 37˚C.
45
3. Place the Ca2+-containing modified Krebs solution, the loading medium, the efflux medium (with or without additions), and the SDS in their appropriate holders at 25˚C.
4. Fill racks with scintillation vials and add 7 mL of scintillation liquid at room temperature to each vial. We perform a 45Ca2+-efflux experiment in each of the 12 wells of cell-culture plate. We add and remove efflux medium 10 times and finally collect the remaining radioactivity. Therefore, 132 scintillation vials are needed.
5. Transfer the 12-well plate with the cells in culture medium from an incubator to the thermostatic plate at 25˚C on a mechanical shaker. 6. Remove the culture medium with a pipette tip connected to a vacuum pump.
The use of a vacuum pump to aspirate the medium instead of a pipette increases the speed of the procedure. See Troubleshooting.
7. Wash each well once with 1 mL of Ca2+-containing modified Krebs solution at 25˚C (i.e., add this medium on top of the cells and immediately remove it with a pipette tip connected to a vacuum pump). See Troubleshooting.
8. Add 1 mL of loading medium to each well at 25˚C to load the stores in the cells with 45Ca2+, and incubate for sufficient time to obtain a steady-state loading. Gently shake the cells. Users should familiarize themselves with how to handle radioactive chemicals. Steady-state loading is usually reached after 1 h, but should be determined empirically. See Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca2+ (Missiaen et al. 2014).
9. Remove the loading medium with a pipette tip connected to a vacuum pump. 10. Wash each well once or twice with 1 mL of efflux medium at 25˚C.
11. Add 0.5-1 mL of efflux medium to each well at 25˚C. Gently shake the cells.
12. After 2 min, remove the efflux medium with a pipette and transfer it to a scintillation vial. See Troubleshooting.
13. Repeat Steps 11 and 12. Because the efflux medium is removed and replaced by new medium each time, it is possible to change its composition (e.g., by adding and subsequently removing an extracellular agonist coupled to IP3 production). The duration of the efflux can be variable, but we normally take samples for 20 min. Use a different pipette for removing the efflux medium and for subsequently adding new efflux medium.
14. At the end of the efflux, add 1 mL of a solution with SDS at 25˚C for 30 min to solubilize all 45 Ca2+ remaining in the stores. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
285
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T. Luyten et al.
15. Remove the solution with a pipette tip and transfer it to a scintillation vial. 16. Count the radioactivity in a scintillation counter. TROUBLESHOOTING Problem (Step 6): Adherent cells are sucked up during the removal of medium. Solution: Never touch the cells on the bottom of the well with the pipette tip. We remove the medium
by tilting the 12-well plate during medium removal and by placing the pipette tip against the lateral wall about 2 mm above the bottom. See Figure 1 in Measurement of Intracellular Ca2+ Release in Permeabilized Cells Using 45Ca2+ (Luyten et al. 2014). Problem (Step 7): The cells detach. Solution: This protocol can only be applied to cells that adhere well to the 12-well cell-culture plates.
Some cell types detach during the various washing steps. One must therefore check whether the cells remain fixed during the whole procedure. One can detect the loss of cells by visual inspection of the remaining monolayer during the flux, by phase-contrast microscopy at the end of the efflux before adding the SDS, or by a sudden noninduced increase in the rate of 45Ca2+ efflux caused by the detachment of radioactive cells. Visual inspection is only possible for some cells like COS-1 cells. Other cells like HeLa cells are less visible and still other cells like ciPTEC cells (conditionally immortalized proximal-tubule epithelial cells) are not visible at all by the naked eye. It sometimes helps one to plate the cells in wells coated with e.g., collagen, poly-L-lysine or gelatin. Problem (Step 12): There is splashing of radioactivity outside the scintillation vial, or the vials turn
sideways in the rack, losing their contents. Solution: Both problems can be avoided by filling the vials with scintillation fluid before starting
the efflux. DISCUSSION
This 45Ca2+-efflux experiment (with the exception of the counting of the radioactivity) takes maximally 2 h. Basic procedures of cell culturing and working with radioactivity are not covered and it is assumed that the reader is familiar with them. For a detailed discussion of this technique see Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca 2+ (Missiaen et al. 2014). The efflux experiment is performed in the absence of extracellular Ca2+ to exclude the contribution of the influx of Ca2+ across the plasma membrane. The agonist-induced increase in the rate of 45 Ca2+ extrusion depends on the activation of the PMCA Ca2+ pumps and of the Na+–Ca2+ exchangers by the increased [Ca2+]cyt resulting from the various Ca2+ fluxes in and out of the various stores. Therefore, a limitation of the technique is that the signal is only a very indirect measure of the intracellular Ca2+ release. It is furthermore impossible in intact cells to eliminate the contribution of some organelles like the mitochondria, because they are needed to produce the ATP that fuels the Ca2+ pumps. The major advantage of experiments in intact cells is that the IP3R can be studied in its natural environment in the presence of all known or unknown regulators of the channel and with feedback of released Ca2+ on its own release. This technique is described for the sake of being comprehensive, but is not in frequent current use. It can be useful in studies of intracellular Ca2+ release where the use of cytosolic Ca2+ dyes is less obvious, for example, when a Ca2+-releasing agent like caffeine interferes with the fluorescence (Van Acker et al. 2004) or when changes in extracellular tonicity occur (Missiaen et al. 1996). Cytosolic Ca2+ dyes are less useful in the latter condition because osmotic fluxes of H2O across the plasma membrane change the dye concentration and the ionic composition in the cell and therefore interfere with the emitted fluorescence. 286
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on June 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Measuring Ca 2+ Release in Intact Cells
RECIPES Ca2+-Containing Modified Krebs Solution
Reagent
Final concentration
NaCl KCl MgCl2 HEPES Glucose CaCl2
135 mM 5.9 mM 1.2 mM 11.6 mM 11.5 mM 1.5 mM
Dissolve chemicals in distilled water and adjust the pH to 7.3 with 10 M NaOH. Store at 4˚C for 1 mo maximum. Efflux Medium for Intact Cells
Reagent
Final concentration
NaCl KCl MgCl2 HEPES Glucose EGTA a A23187 (2 mM in ethanol) a Thapsigargin (2 mM in ethanol)
135 mM 5.9 mM 1.2 mM 11.6 mM 11.5 mM 2 mM 10 µM 2 µM
Dissolve all chemicals in distilled water and adjust the pH to 7.3 with 10 M NaOH. Store at 4˚C for 1 mo maximum. a
Optional agents that can be added to the standard efflux medium.
Loading Medium for Intact Cells
Reagent NaCl KCl MgCl2 HEPES Glucose 40 CaCl2 + 45CaCl2
Final concentration 135 mM 5.9 mM 1.2 mM 11.6 mM 11.5 mM 0.2 mM
Dissolve chemicals in distilled water and adjust the pH to 7.3 with 10 M NaOH. Store medium (without 45CaCl2) at 4˚C for 1 mo maximum.
The 45Ca2+ should have a specific activity of 0.5 MBq/mL. For example, use 14 µM 45Ca2+ from PerkinElmer (NEZ013; specific activity of 802.65 MBq/mg; concentration of 1465.77 MBq/mL) and 186 µM 40Ca2+.
ACKNOWLEDGMENTS
We thank Marina Crabbe, Anja Florizoone, and Kirsten Welkenhuyzen for excellent technical help. This work was supported by the Research Council of the KU Leuven via the Concerted Actions Program (GOA/09/012) and by the Interuniversity Attraction Poles Program (Belgian Science Policy; P6/28). Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
287
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on June 11, 2015 - Published by Cold Spring Harbor Laboratory Press
T. Luyten et al.
REFERENCES Luyten T, Bultynck G, Parys JB, De Smedt H, Missiaen L. 2014. Measurement of intracellular Ca2+ release in permeabilized cells using 45Ca2+. Cold Spring Harb Protoc doi: 10.1101/pdb.prot073189. Missiaen L, De Smedt H, Parys JB, Sienaert I, Vanlingen S, Droogmans G, Nilius B, Casteels R. 1996. Hypotonically induced calcium release from intracellular calcium stores. J Biol Chem 271: 4601–4604. Missiaen L, Luyten T, Bultynck G, Parys JB, De Smedt H. 2014. Measurement of intracellular Ca2+ release in intact and permeabi-
288
lized cells using 45Ca2+. Cold Spring Harb Protoc doi: 10.1101/pdb. top066126. Van Acker K, Bultynck G, Rossi D, Sorrentino V, Boens N, Missiaen L, De Smedt H, Parys JB, Callewaert G. 2004. The 12 kDa FK506-binding protein, FKBP12, modulates the Ca2+-flux properties of the type-3 ryanodine receptor. J Cell Sci 117: 1129–1137.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073197
Downloaded from http://cshprotocols.cshlp.org/ at The University of Iowa Libraries on June 11, 2015 - Published by Cold Spring Harbor Laboratory Press
Measurement of Intracellular Ca2+ Release in Intact Cells Using 45Ca2+ Tomas Luyten, Geert Bultynck, Jan B. Parys, Humbert De Smedt and Ludwig Missiaen Cold Spring Harb Protoc; doi: 10.1101/pdb.prot073197 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 (1115 articles) Signal Transduction (55 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
