We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.
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Method Article
Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes
In This Article
Summary
Abstract
In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.
Protocol
1. Preparation of Resting Human T Lymphocytes
- Using RosetteSep Human T Cell Enrichment Cocktail and RosetteSep Density Medium, purify T cells from human blood samples according to the manufacturer's instructions. The resulting cell population should contain 95% CD3+ resting T cells. We purify human T lymphocytes from peripheral blood samples collected from healthy volunteers in accordance with the protocol approved by the UC Davis Internal Review Board.
- After isolation, resuspend resting T cells at a density of 0.5 x 106 cells/ mL in cell culture medium containing RPMI-1640 medium with glutamine and HEPES, supplem....
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Discussion
The electrophysiological investigation of CRAC currents in resting human T cells is a challenging task because the endogenous CRAC current amplitude in these cells is small due to the small cell size (the resting human T cell diameter is in the range of 5-8 μm). Here, we present a step-by-step procedure to reliably record CRAC currents in resting human T lymphocytes isolated from peripheral blood mononuclear cells. This technology allows us to investigate the physiology and functional expression of CRAC channels in re.......
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Disclosures
No conflicts of interest declared.
Acknowledgements
We are thankful to the Department of Physiology and Membrane Biology, University of California Davis for providing us with facilities and an excellent environment for the studies of ion channels.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| RosetteSep Human T Cell Enrichment Cocktail | Stem Cell Technologies | 15061 | |
| RosetteSep Density Medium | Stem Cell Technologies | 15705 | |
| RPMI-in 1640 medium w/glutamine/HEPES | Fisher Scientific | SH3025501 | |
| Fetal Calf Serum | Omega Scientific | FB-01 | |
| GlutaMAX-I (100X solution) | Invitrogen | 35050 | |
| RPMI 1640 vitamin solution (100X) | Sigma-Aldrich | 7256 | |
| 1640 amino acids solution (50X) | Sigma-Aldrich | R7131 | |
| Sodium pyruvate | Sigma-Aldrich | S8636 | |
| β-Mercapt–thanol | Sigma-Aldrich | M7522 | |
| Inositol trisphosphate | Sigma-Aldrich | 19766 | |
| BAPTA | Sigma-Aldrich | A4926 | |
| Poly-L-Lysine Hydrobromide | Sigma-Aldrich | P2636 | |
| Lanthanum Chloride | Sigma-Aldrich | 262072 | |
| Thapsigargin | Calbiochem | 586005 | |
| Sylgard 184 Silicon Elastomer Kit | Dow Corning | 3097358-1004 | |
| HIPEC R6101 Semiconductor Protective Coating | Dow Corning | ||
| 63-500 Series High-Performance Vibration Isolation Lab Table | Technical Manufacturing Corp. | 63-540 | |
| EPC 10 patch clamp amplifier with headstage | HEKA Instruments | ||
| Micromanipulator | Sutter Instrument Co. | MP-285 | |
| Olympus 1X71 Inverted microscope with 40x oil immersion objective | Olympus Corporation | 1X71 | |
| Windows Computer | Dell | ||
| Pulse software | HEKA Instruments | ||
| Origin Scientific Graphing and Analysis Software | OriginLab | ||
| Patch pipette puller | Sutter Instrument Co. | P-97 | |
| Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm | Sutter Instrument Co. | BF150-110-7.5 | |
| Narashige’s Microforge | Tritech Research, Inc. | MF-830 | |
| Silicon O-rings | McMaster-Carr | 111 S70 | |
| Coverslips 25 mm | Fisher Scientific | 12-545-102 25 mm 25CIR.-1 |
References
- Parekh, A. B. Store-operated CRAC channels: function in health and disease. Nat Rev Drug Discov. 9, 399-410 (2010).
- Feske, S. CRAC channelopathies. Pflugers Arch. , (2010).
- Lewis, R. S., Cahalan,
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