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我们采用荧光钙指示剂喹2,建立了测定人血小板胞浆游离钙浓度的方法,简报如下。 导入喹2的血小板悬液(QPS)的制备:将富含血小板血浆与10 μmol/L 喹2/AM混匀,置22℃水浴20 min,加入1μmol/L PGE_1,室温下430×g 离心 15 min,弃上层液。用1ml含1 μmol/L PGE_1和10μmol/L EGTA的HEPES无钙台氏缓冲液(HTB)使血小板悬浮并移至10ml体积已用HTB(含10 μmol/L EGTA)50ml平衡过的2％琼脂糖胶柱上,用相同缓冲液洗脱。过柱后将血小板悬液调至1×10~8血小板/ml和Ca~(2+)浓度至1 mmol/L,置22℃备用。对照血小板悬液(CPS)的制备:用等体积
We use fluorescent calcium indicator Quin 2, established a method of determination of human platelet cytoplasm free calcium concentration, the briefing is as follows. Preparation of QuQ2-loaded platelet suspension (QPS): The platelet-rich plasma was mixed with 10 μmol / L Qua2 / AM for 20 min at 22 ° C, 1 μmol / L PGE_1 added, 430 × g centrifuged at room temperature min, discard the supernatant. Platelets were suspended and transferred to 10 ml volumes of 2% agar that had been equilibrated with 50 ml of HTB (containing 10 μmol / L EGTA) with 1 ml of HEPES calcium-free Tyrode’s buffer (HTB) containing 1 μmol / L PGE_1 and 10 μmol / On a gum column, elute with the same buffer. After the column was adjusted to platelet suspension 1 × 10 ~ 8 platelet / ml and Ca ~ (2+) concentration to 1 mmol / L, set at 22 ℃ for use. Preparation of Control Platelet Suspension (CPS): Equal volumes were used