Protocol

The Minimal Requirements to Use Calcium Imaging to Analyze ICRAC Dalia Alansary,1 Tatiana Kilch,1 Christian Holzmann, Christine Peinelt, Markus Hoth, and Annette Lis2 Department of Biophysics, Saarland University, Homburg, Germany

Endogenous calcium release-activated channel (CRAC) currents are usually quite small and not always easy to measure using the patch-clamp technique. While we have, for instance, successfully recorded very small CRAC currents in primary human effector T cells, we have not yet managed to record CRAC in naïve primary human T cells. Many groups, including ours, therefore use Ca2+ imaging technologies to analyze CRAC-dependent Ca2+ influx. However, Ca2+ signals are quite complex and depend on many different transporter activities; thus, it is not trivial to make quantitative statements about one single transporter, in this case CRAC channels. Therefore, a detailed patch-clamp analysis of ICRAC is always preferred. Since many laboratories use Ca2+ imaging for ICRAC analysis, we detail here the minimal requirements for reliable measurements. Ca2+ signals not only depend on the net Ca2+ influx through CRAC channels but also depend on other Ca2+ influx mechanisms, K+ channels or Cl− channels (which determine the membrane potential), Ca2+ export mechanisms like plasma membrane Ca2+ ATPase (PMCA), sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) or Na+–Ca2+ exchangers, and (local) Ca2+ buffering often by mitochondria. In this protocol, we summarize a set of experiments that allow (quantitative) statements about CRAC channel activity using Ca2+ imaging experiments, including the ability to rule out Ca2+ signals from other sources.

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 article for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.

Reagents

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 1 µM) in ethanol Cell culture medium Cell line of choice Extracellular solution with 1 mM Ca2+ Extracellular solution with no free Ca2+ Fura-2 AM (1 µM) in dimethyl sulfoxide (DMSO), or other preferentially ratiometric dyes like Indo-1 (use for measurements at room temperature; see Step 3) Fura-PE3 AM (1 µM) in DMSO (use for measurements at 37˚C; see Step 3) HEPES (10–20 mM; optional, see Step 1) 1

These authors contributed equally. Correspondence: [email protected]

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Using Calcium Imaging to Analyze I CRAC

Poly-L-lysine hydrobromide (0.1 mg/mL) in water Poly-L-ornithine hydrobromide (0.1 mg/mL) in water Thapsigargin (1 µM) in DMSO Alternatively, cyclopiazonic acid (CPA; 30 µM) or 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ; 30 µM) can be used.

Equipment

Imaging setup Use a standard Ca2+ imaging setup for single-cell analysis with an UV optimized light path and objective in case UV dyes like Fura-2 or Indo-1 are used. Use 20× or 40× objectives (depending on cell size) with optimized UV efficiency (for Fura-2 or Indo-1). Use a small measuring chamber that is optimized for solution exchange. We use a self-made sandwich chamber that accommodates 60 µL of solution and can be completely exchanged within 1 sec.

METHOD Measuring CRAC Activity

1. Load cells with Fura-2 AM at room temperature (and not at 37˚C) for the shortest time possible that is still sufficient to obtain a good fluorescent signal over background.

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Keep in mind that the concentration of Fura-2 and loading time vary by cell type. For Jurkat cells, we use 1 µM Fura-2 AM (in DMSO) for 15 min, and for primary human T cells transfected with siRNA, we use 2 µM Fura-2 AM for 30 min. Load 0.5–1.0 million nonadherent T cells in 1 mL culture medium (the pH should be buffered with 10–20 mM HEPES if there is no incubator at room temperature) or in 1 mL extracellular solution with 1 mM Ca2+. For adherent RBL cells, load the cells in their chamber, which should have a glass bottom for microscopy. Do not load at 37˚C to avoid a large accumulation of Fura-2 in cell organelles like mitochondria and endoplasmic reticulum (Quintana and Hoth 2004), which may be a problem in many cell types. Many groups use pluronic acid to facilitate FURA-2 AM dye loading; we have observed neither advantages nor disadvantages in T cells or mast cells.

2. Wash cells at least twice with extracellular solution containing 1 mM Ca2+ as carefully as possible. 3. Wait 15 min to allow cleavage of the Fura-AM. Incubation can be performed at room temperature or at 37˚C, but cells should not be left at 37˚C for longer than 15 min, because Fura-2 is transported out of many cell types. If experiments are to be performed at 37˚C, use Fura-PE3 (Dolmetsch et al. 1998), which must be loaded for 2 h but does not leak out of cells as quickly. 4. For nonadherent T cells, coat the measuring chamber with a glass bottom and coverslip with 0.1 mg/mL polylysine or polyornithine for 30 min (before or during Fura-2 loading). Suck the remaining polylysine or polyornithine off and rinse the coverslip with water and air-dry. 5. Place the cells on the coverslip and allow them to adhere for 5 min. Assemble the sandwich chamber and wash with 500 µL of 1 mM Ca2+ extracellular solution. Avoid light exposure to prevent light-induced activation or damage of cells. Start the measurement with a baseline recording. If loading and washing of the cells was optimal, cells should not show elevated cytosolic Ca 2+ concentrations.

6. Measure CRAC activity as follows: i. Apply extracellular solution with no free Ca2+ and 1 µM thapsigargin (or 30 µM CPA or tBHQ) for 10 min to deplete Ca2+ stores. ii. Replace solution with extracellular solution with 1 mM Ca2+ and 1 µM thapsigargin to access net store-operated Ca2+ influx for at least 10 min until a steady-state cytosolic Ca2+ concentration is reached. iii. Replace solution with extracellular solution with no free Ca2+ and 1 µM thapsigargin to access Ca2+ export kinetics. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073262

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D. Alansary et al. A typical experiment is shown in Figure 1. Unopposed leakage of Ca 2+ from the stores results in a first transient peak, which is followed by the Ca 2+ influx through store-operated channels. From this experiment, it is possible to determine the net Ca 2+ entry into the cytosol. Net Ca 2+ entry correlates best with the initial Ca 2+ influx rate (which can be analyzed following differentiation of the curve) or the steadystate Ca 2+ plateau but not in all instances with the Ca 2+ peak. The amplitude of the Ca 2+ peak depends largely on CRAC channel inactivation and PMCA up-regulation in T cells (Bautista et al. 2002; Hoth et al. 1997) and may not always reflect the amplitude of ICRAC. Calibration of the ratiometric Fura-2 signals is performed according to Grynkiewicz et al. (1985). See Troubleshooting.

Testing the Effects of Blockers A typical question in CRAC channel research is how to test if a change in the cytosolic Ca 2+ concentration (for instance by a blocker) is directly related to CRAC channel activity. As mentioned before, this question is best answered using the patch clamp technique, but if CRAC currents are too small, can also be tested by Ca 2+ imaging as described here (compare also Zitt et al. [2004]). If a blocker is applied that reduces the cytosolic Ca 2+ concentration, the reduction could in principle be caused by an effect on CRAC itself, on K + or Cl − channels (and a change in membrane potential and driving force for CRAC Ca 2+ entry), a change in the Ca 2+ export rate, or a change in Ca 2+ buffering by mitochondria, which strongly modulates CRAC activity (Bautista et al. 2002; Hoth et al. 1997; Quintana et al. 2006; Zitt et al. 2004). The following experiments are designed to test if the blocker directly inhibits CRAC channels or other mechanisms that influence cytosolic Ca 2+ signals.

7. To test membrane potential and K+ channels, repeat Step 6, exchanging KCl for NaCl in the extracellular solution. Doing this will hold the membrane potential around 0 mV and make the Ca 2+ measurements independent of K + channel activity, because the external K + concentration matches the internal one. Under these conditions, there is no net K + current and the membrane potential does not deviate much from 0 mV regardless if K + channels are active or not (in case the blocker interferes with K + channels). As the driving force for entry through CRAC channels is reduced at 0 mV, the extracellular Ca 2+ concentration has to be increased to for instance 3 mM in T cells (Fasolato et al. 1993; Zitt et al. 2004) and potentially to even higher concentrations in cells with smaller Ca 2+ influx. If the reduction of the cytosolic Ca 2+ concentration by the blocker is still present, a potential blockade of K + channels does not explain the observed effect.

8. To test the role of CRAC modulation by mitochondria, repeat Step 6, adding 1 µM CCCP to the extracellular solution to inhibit mitochondrial Ca2+ uptake, buffering, and modulation of CRAC currents (Hoth et al. 1997; Quintana et al. 2006). As Ca 2+ entry through CRAC channels is reduced by inhibition of mitochondrial Ca 2+ uptake, the extracellular Ca 2+ concentration has to be increased to for instance 5 mM. If the reduction of the cytosolic Ca 2+ concentration by the blocker is still present, mitochondrial Ca 2+ uptake does not explain the observed effect.

9. Test calcium export.



Measure Ca2+ export under the different conditions (with and without an inhibitor) and compare the rates of cytosolic Ca2+ decline after switching the solution back from 1 mM Ca2+ to Ca2+-free extracellular solution (Fig. 1). If the rates are the same, Ca 2+ export mechanisms most likely are not changed by the blocker.

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FIGURE 1. A representative course of a typical Ca2+ re2+ addition protocol. The upper bar shows the [Ca ]o and the trace shows corresponding alterations in [Ca2+]i of Fura-2-loaded cells.

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Using Calcium Imaging to Analyze I CRAC



An alternative, preferred experiment is to measure Ca2+ clearance rates in combined patchimaging experiments at low buffering conditions when ICRAC can be measured simultaneously with Ca2+ signals in T cells (Zweifach and Lewis 1995; Hoth et al. 1997; Bautista et al. 2002). Low buffering conditions have also been applied extensively to analyze ICRAC in mast cells (Fierro and Parekh 2000; Glitsch et al. 2002).

10. If in Steps 7, 8, and 9 a blocker effect is still visible, the blocker acts most likely directly on CRAC channels. Steps 7–9 can be combined in one experiment to further test this statement. If another store-operated Ca2+ influx mechanism is present besides CRAC channels, this can only be separated using pharmacological tools (see Measuring Endogenous ICRAC and ORAI Currents with the Patch Clamp Technique [Alansary et al. 2014]); however, in many cell types, CRAC channels appear to dominate store-operated Ca2+ influx.

TROUBLESHOOTING Problem (Step 6): Ca2+ loading of the single cells is very heterogeneous. Solution: Thoroughly vortex dye before diluting the cells into it. We also use a very slowly rotating

wheel during incubation to avoid accumulation of the cells at one spot caused by gravity. Problem (Step 6): Kinetics of the cytosolic Ca2+ changes are slow and Ca2+ signals are small. Solution: Check Fura-2 loading. Too much Fura-2 in the cells will buffer cytosolic Ca2+ dramatically,

slow kinetics, and dampen Ca2+ signals. Decrease Fura-2 loading.

Problem (Step 6): Cells are preactivated or become activated by light (i.e., cytosolic Ca2+ is already

elevated at the beginning of the experiment). Solution: Treat cells more gently, decrease excitation of Fura-2 time as much as possible, always close

the shutter between sampling the cells, and try to avoid very low UV light (

The minimal requirements to use calcium imaging to analyze ICRAC.

Endogenous calcium release-activated channel (CRAC) currents are usually quite small and not always easy to measure using the patch-clamp technique. W...
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