Pollack V A, Brandhorst J S, Hanna M G
J Immunol Methods. 1981;41(1):29-41. doi: 10.1016/0022-1759(81)90271-4.
We present a simple method for the recovery of lymphocytes from guinea pig peripheral blood by discontinuous density gradient centrifugation using Ficoll-metrizoate solutions. This technique is formulated specifically for the routine preparation of guinea pig lymphocytes for in vitro cultivation and is capable of recovering 40-60% of available lymphocytes. Whole, heparinized (10-100 U/ml) blood was drawn from strain 2 guinea pigs by cardiac puncture, diluted 1 : 4 with Ca2,Mga free Hanks' balanced salt solution (CMF-HBSS) containing EDTA (5 mM) and gentamicin (50 microgram/ml), and incubated at room temperature for 30-60 min to promote leukocyte disaggregation. Five volumes of diluted blood were layered onto 2 vol of Ficoll-metrizoate adjusted to a density of 1.107 g/ml with sodium metrizoate solution. A band of mononuclear cells (80-90% lymphocytes, 10-20% monocytes, and less than 2% granulocytes) formed at the interface after centrifugation (400 X g, 20-40 min, room temperature). More than 95% of the cells were viable by trypan blue exclusion. Lymphocytes recovered from as little as 3-5 ml whole blood were more sensitive to antigen- or mitogen-activated transformation than leukocyte suspensions obtained by dextran-citrate sedimentation with or without nylon column filtration.
我们提出了一种通过使用菲可-泛影酸盐溶液进行不连续密度梯度离心从豚鼠外周血中分离淋巴细胞的简单方法。该技术是专门为常规制备用于体外培养的豚鼠淋巴细胞而设计的，能够回收40%-60%的可用淋巴细胞。通过心脏穿刺从2系豚鼠中采集全血并肝素化（10-100 U/ml），用含有乙二胺四乙酸（5 mM）和庆大霉素（50微克/毫升）的无钙、镁的汉克斯平衡盐溶液（CMF-HBSS）按1:4稀释，并在室温下孵育30-60分钟以促进白细胞解聚。将五倍体积的稀释血液铺在两体积用泛影酸钠溶液调节至密度为1.107克/毫升的菲可-泛影酸盐上。离心（400×g，20-40分钟，室温）后，在界面处形成一条单核细胞带（80%-90%为淋巴细胞，10%-20%为单核细胞，粒细胞少于2%）。通过台盼蓝拒染法检测，超过95%的细胞具有活力。从仅3-5毫升全血中回收的淋巴细胞比通过右旋糖酐-柠檬酸盐沉降法（有无尼龙柱过滤）获得的白细胞悬液对抗原或丝裂原激活的转化更敏感。
