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Protocol
Measurement of Intracellular Ca2+ Release in Permeabilized 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 to measure Ca2+ release from the nonmitochondrial intracellular Ca2+ stores in monolayers of saponin-permeabilized cells cultured in 12-well 4-cm2 clusters. The 45 Ca2+-flux technique described here can only be applied to cell types that still adhere to the plastic after exposing them to saponin. We describe the permeabilization procedure, the loading of the nonmitochondrial Ca2+ 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
Cell line of interest and appropriate culture medium (see Step 1) Efflux medium for permeabilized cells, freshly prepared Prepare efflux medium with appropriate additions. See Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca2+ (Missiaen et al. 2014).
Loading medium for permeabilized cells, freshly prepared S− and S+ media, freshly prepared 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 Cell-culture incubator Vacuum pump 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073189
289
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF ALABAMA AT BIRMINGHAM/ LISTER HILL LIBRARY OF THE HEALTH SCIENCES on July 26, 2015 - Published by Cold Spring Harbor Laboratory Press
T. Luyten et al.
METHOD
1. Plate the 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 30˚C, and start the circulation to the thermostatic plate and the holders of the various solutions. 3. Place freshly prepared efflux medium (with or without additions), loading medium, S− medium, S+ medium, and SDS in their appropriate holders at 30˚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 9 times and finally collect the remaining radioactivity. Therefore 120 scintillation vials are needed.
5. Transfer the 12-well plate with the cells in culture medium from an incubator to the thermostatic plate at 30˚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 S− medium at 30˚C (i.e., add S− medium on top of the cells and immediately remove it with a pipette tip connected to a vacuum pump). 8. Add 1 mL of S+ medium to each well at 30˚C. Incubate for 10 min.
9. Remove the S+ medium with a pipette tip connected to a vacuum pump. Insufficient removal of saponin can lead to further permeabilization during loading of the cells with 45Ca2+. Therefore, after removing the S+ medium from the wells with a pipette tip connected to a vacuum pump, we include a second step of aspiration of the remaining S+ medium. See Troubleshooting.
10. Wash each well once with 1 mL of S− medium at 30˚C.
11. Add 1 mL of loading medium to each well at 30˚C to load the stores with 45Ca2+, and incubate for sufficient time to obtain a steady-state loading. Users should familiarize themselves with how to handle radioactive chemicals. Steady-state loading is usually reached after 45 min, but should be determined empirically. See Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca2+ (Missiaen et al. 2014).
12. Remove the loading medium with a pipette tip connected to a vacuum pump. 13. Wash each well once or twice with 1 mL of efflux medium at 30˚C.
14. Add 0.5–1 mL of efflux medium at 30˚C. Gently shake the cells.
15. After 2 min, remove the efflux medium with a pipette and transfer it to a scintillation vial. The medium can also be removed after a shorter time period. If the time periods are very short (e.g., 6 sec), continuously remove the efflux medium with a fraction collector and, once the well is almost empty, replace it manually with 1 mL of new efflux medium. The latter technique can only be done on one well at a time, and allows for testing a concentration ramp of IP3. See Troubleshooting.
16. Repeat Steps 14 and 15. 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 IP3 or a Ca2+ ionophore). The duration of the efflux can be variable, but we normally take samples for 18 min. Use a different pipette for removing the efflux medium and for subsequently adding new efflux medium.
290
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073189
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF ALABAMA AT BIRMINGHAM/ LISTER HILL LIBRARY OF THE HEALTH SCIENCES on July 26, 2015 - Published by Cold Spring Harbor Laboratory Press
Measuring Ca 2+ Release in Permeabilized Cells
17. At the end of the efflux, add 1 mL of 2% SDS at 30˚C for 30 min to solubilize all 45Ca2+ remaining in the stores. 18. Remove the solution with a pipette tip and transfer it to a scintillation vial. 19. 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 2 mm above the bottom (Fig. 1). Problem (Step 9): 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, especially after permeabilization with saponin. One must therefore check whether the cells remain well attached during the whole procedure. The loss of cells can be detected 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 to plate cells on wells coated with e.g., collagen, poly-Llysine, or gelatin. Problem (Step 15): 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 liquid before starting
the efflux.
DISCUSSION
This 45Ca2+-efflux experiment (with the exception of the counting of the radioactivity) takes maximally 3 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 45Ca2+ (Missiaen et al. 2014). The major advantages of this 45Ca2+-efflux technique in plasma-membrane permeabilized cells are (i) that unidirectional 45Ca2+ effluxes are measured, (ii) that the conditions at the cytosolic side of the channel can be exactly controlled because of the direct access to the internal stores, and (iii) the very high accuracy and reproducibility. The major limitations are (i) the slow sampling rate (1 measurement each 2 min, up to 1 measurement each 6 sec), (ii) that the technique does not allow the study of the IP3R in its natural cytosolic environment, (iii) that released Ca2+ cannot exert feedback on its own release because of the presence of a Ca2+ buffer, (iv) that saponin causes a dramatic change in the cell architecture, making the technique unsuitable to study the interaction of the ER with other organelles like the mitochondria, (v) that the technique is not suitable for high-throughput experiments, and (vi) that the relatively large volumes of efflux medium (0.5 to 1 mL) sometimes require large amounts of purified proteins or other compounds to be tested. Some of these limitations can be solved, e.g., superfusion systems on cells or microsomes attached to filters can reach a time resolution
Protocol
Measurement of Intracellular Ca2+ Release in Permeabilized 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 to measure Ca2+ release from the nonmitochondrial intracellular Ca2+ stores in monolayers of saponin-permeabilized cells cultured in 12-well 4-cm2 clusters. The 45 Ca2+-flux technique described here can only be applied to cell types that still adhere to the plastic after exposing them to saponin. We describe the permeabilization procedure, the loading of the nonmitochondrial Ca2+ 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
Cell line of interest and appropriate culture medium (see Step 1) Efflux medium for permeabilized cells, freshly prepared Prepare efflux medium with appropriate additions. See Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca2+ (Missiaen et al. 2014).
Loading medium for permeabilized cells, freshly prepared S− and S+ media, freshly prepared 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 Cell-culture incubator Vacuum pump 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073189
289
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF ALABAMA AT BIRMINGHAM/ LISTER HILL LIBRARY OF THE HEALTH SCIENCES on July 26, 2015 - Published by Cold Spring Harbor Laboratory Press
T. Luyten et al.
METHOD
1. Plate the 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 30˚C, and start the circulation to the thermostatic plate and the holders of the various solutions. 3. Place freshly prepared efflux medium (with or without additions), loading medium, S− medium, S+ medium, and SDS in their appropriate holders at 30˚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 9 times and finally collect the remaining radioactivity. Therefore 120 scintillation vials are needed.
5. Transfer the 12-well plate with the cells in culture medium from an incubator to the thermostatic plate at 30˚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 S− medium at 30˚C (i.e., add S− medium on top of the cells and immediately remove it with a pipette tip connected to a vacuum pump). 8. Add 1 mL of S+ medium to each well at 30˚C. Incubate for 10 min.
9. Remove the S+ medium with a pipette tip connected to a vacuum pump. Insufficient removal of saponin can lead to further permeabilization during loading of the cells with 45Ca2+. Therefore, after removing the S+ medium from the wells with a pipette tip connected to a vacuum pump, we include a second step of aspiration of the remaining S+ medium. See Troubleshooting.
10. Wash each well once with 1 mL of S− medium at 30˚C.
11. Add 1 mL of loading medium to each well at 30˚C to load the stores with 45Ca2+, and incubate for sufficient time to obtain a steady-state loading. Users should familiarize themselves with how to handle radioactive chemicals. Steady-state loading is usually reached after 45 min, but should be determined empirically. See Measurement of Intracellular Ca2+ Release in Intact and Permeabilized Cells Using 45Ca2+ (Missiaen et al. 2014).
12. Remove the loading medium with a pipette tip connected to a vacuum pump. 13. Wash each well once or twice with 1 mL of efflux medium at 30˚C.
14. Add 0.5–1 mL of efflux medium at 30˚C. Gently shake the cells.
15. After 2 min, remove the efflux medium with a pipette and transfer it to a scintillation vial. The medium can also be removed after a shorter time period. If the time periods are very short (e.g., 6 sec), continuously remove the efflux medium with a fraction collector and, once the well is almost empty, replace it manually with 1 mL of new efflux medium. The latter technique can only be done on one well at a time, and allows for testing a concentration ramp of IP3. See Troubleshooting.
16. Repeat Steps 14 and 15. 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 IP3 or a Ca2+ ionophore). The duration of the efflux can be variable, but we normally take samples for 18 min. Use a different pipette for removing the efflux medium and for subsequently adding new efflux medium.
290
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073189
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF ALABAMA AT BIRMINGHAM/ LISTER HILL LIBRARY OF THE HEALTH SCIENCES on July 26, 2015 - Published by Cold Spring Harbor Laboratory Press
Measuring Ca 2+ Release in Permeabilized Cells
17. At the end of the efflux, add 1 mL of 2% SDS at 30˚C for 30 min to solubilize all 45Ca2+ remaining in the stores. 18. Remove the solution with a pipette tip and transfer it to a scintillation vial. 19. 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 2 mm above the bottom (Fig. 1). Problem (Step 9): 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, especially after permeabilization with saponin. One must therefore check whether the cells remain well attached during the whole procedure. The loss of cells can be detected 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 to plate cells on wells coated with e.g., collagen, poly-Llysine, or gelatin. Problem (Step 15): 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 liquid before starting
the efflux.
DISCUSSION
This 45Ca2+-efflux experiment (with the exception of the counting of the radioactivity) takes maximally 3 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 45Ca2+ (Missiaen et al. 2014). The major advantages of this 45Ca2+-efflux technique in plasma-membrane permeabilized cells are (i) that unidirectional 45Ca2+ effluxes are measured, (ii) that the conditions at the cytosolic side of the channel can be exactly controlled because of the direct access to the internal stores, and (iii) the very high accuracy and reproducibility. The major limitations are (i) the slow sampling rate (1 measurement each 2 min, up to 1 measurement each 6 sec), (ii) that the technique does not allow the study of the IP3R in its natural cytosolic environment, (iii) that released Ca2+ cannot exert feedback on its own release because of the presence of a Ca2+ buffer, (iv) that saponin causes a dramatic change in the cell architecture, making the technique unsuitable to study the interaction of the ER with other organelles like the mitochondria, (v) that the technique is not suitable for high-throughput experiments, and (vi) that the relatively large volumes of efflux medium (0.5 to 1 mL) sometimes require large amounts of purified proteins or other compounds to be tested. Some of these limitations can be solved, e.g., superfusion systems on cells or microsomes attached to filters can reach a time resolution
