在人T淋巴细胞的全细胞钙释放激活的钙记录（CRAC）的电流
Summary
钙离子释放活化钙全细胞膜片钳记录（CRAC）外周血单核细胞衍生的人类T淋巴细胞中的电流，我们提供了一步一步的协议。
Abstract
T淋巴细胞，耗尽的Ca 2 +从细胞内Ca 2 +存储信息激活质膜钙离子通道，被称为钙释放激活钙（CRAC）通道。 CRAC通道中的T细胞增殖和基因表达调控中发挥重要作用。 T细胞异常的CRAC通道的功能已被链接到重症联合免疫缺陷和自身免疫性疾病1，2。在人类T细胞研究的CRAC通道功能调节正常的免疫反应，可能会发现新的分子机制，并揭开有关人类疾病的原因。膜电流的电生理记录功能通道特性及其调控提供最准确的评估。然而，在正常的人体T细胞的CRAC电流测量，人类白血病T细胞株，Jurkat T细胞的CRAC通道电流的电生理评估是第一次执行超过20年，前3仍然是一个具有挑战性的任务。在录音中正常T细胞的CRAC通道电流的困难加剧的事实，血源性T淋巴细胞Jurkat T细胞的大小比要小得多，因此，全细胞内源性的CRAC电流幅度非常低。在这里，我们给一步一步的过程，我们经常使用记录的 Ca 2 +或Na +通过在休息从健康志愿者的外周血中分离的人类T细胞的CRAC通道的电流。这里描述的方法记录在Jurkat T细胞的CRAC电流激活人体T细胞4-8所使用的程序获得通过。
Protocol
1。准备休息的人T淋巴细胞使用RosetteSep人类T细胞富集鸡尾酒和RosetteSep密度中等，净化T细胞从人体血液样本，根据制造商的指示。由此产生的细胞群，应含有95％的CD3 +静止T细胞。我们净化人类T淋巴细胞，从外周血中，从健康志愿者按照由加州大学戴维斯分校的内部审查委员会批准的协议采集的样本。 隔离后，重新悬浮静止T细胞在细胞培养液中中含有谷氨酰胺和HEPES的RPMI …
Discussion
人类T细胞在休息的CRAC电流的电生理调查是一项具有挑战性的任务，因为在这些细胞中的内源性的CRAC的电流幅值小，由于小单元尺寸（休息人体T细胞直径在5-8微米的范围内）。在这里，我们现在一步一步的程序，以可靠地记录在休息的人类T淋巴细胞从外周血单个核细胞中分离的CRAC电流。这项技术使我们能够探讨在静息T细胞的CRAC通道的生理学和功能表达，以便更好地了解正常和病理的免疫细胞?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢为我们提供与离子通道的研究设施和良好的环境，加州大学戴维斯分校生理学与膜生物学部。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| RosetteSep Human T Cell Enrichment Cocktail | StemCell Technologies, Vancouver, BC, Canada | 15061 | ||
| RosetteSep Density Medium | StemCell Technologies | 15705 | ||
| RPMI-in 1640 medium w/glutamine/HEPES | Fisher, Waltham, MA | SH3025501 | ||
| Fetal Calf Serum | Omega Scientific, Tarzana, CA | FB-01 | ||
| GlutaMAX-I (100X solution) | Invitrogen, Carlsbad, CA | 35050 | ||
| RPMI 1640 vitamin solution (100X) | Sigma-Aldrich | 7256 | ||
| 1640 amino acids solution (50X) | Sigma-Aldrich | R7131 | ||
| Sodium pyruvate | Sigma-Aldrich | S8636 | ||
| β-Mercaptoethanol | 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, Midland, MI | 3097358-1004 | ||
| HIPEC R6101 Semiconductor Protective Coating | Dow Corning, Midland, MI | |||
| 63-500 Series High-Performance Vibration Isolation Lab Table | Technical Manufacturing, Peabody, MA | 63-540 | ||
| EPC 10 patch clamp amplifier with headstage | HEKA Instruments, Bellmore, NY | |||
| Micromanipulator | Sutter Instrument, Novato, CA | MP-285 | ||
| Olympus 1X71 Inverted microscope with 40x oil immersion objective | Olympus America, Center Valley, PA | 1X71 | ||
| Windows Computer | Dell | |||
| Pulse software | HEKA Instruments | |||
| Origin Scientific Graphing and Analysis Software | OriginLab, Northampton, MA | |||
| Patch pipette puller | Sutter Instrument | P-97 | ||
| Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm | Sutter Instrument | BF150-110-7.5 | ||
| Narashige’s Microforge | Tritech Research, Los Angeles, CA | MF-830 | ||
| Silicon O-rings | McMASTER-CARR, Santa Fe Springs, CA | 111 S70 | ||
| Coverslips 25 mm | Fisher Scientific | 12-545-102 25 mm 25CIR.-1 |
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Tags
Whole-cell RecordingCalcium Release-activated Calcium CurrentsCRAC ChannelsT LymphocytesIntracellular Ca2+ StorePlasmalemmal Ca2+ ChannelsT Cell ProliferationGene ExpressionSevere Combined ImmunodeficiencyAutoimmune DiseasesFunctional Channel PropertiesRegulationJurkat T CellsHuman Leukemia T Cell LineNormal Human T CellsBlood-derived T LymphocytesAmplitudeCa2+ CurrentsNa+ Currents
Citar este artigo
Thakur, P., Fomina, A. F. Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes. J. Vis. Exp. (46), e2346, doi:10.3791/2346 (2010).