Comparison of several methods for the isolation of murine hepatic lymphocytes
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摘要: 目的:通过比较几种肝脏淋巴细胞分离方法得到的总淋巴细胞、T细胞、自然杀伤T细胞(NKT cells)及自然杀伤细胞(NK细胞)的比例和细胞绝对数,明确各种方法的适用范围。方法:按照析因设计,将不放血(A法)、摘眼球放血(B法)、体循环灌流(C法)、摘眼球结合体循环灌流(B法+C法)4种肝脏预处理方法和单浓度离心法(Ⅰ法)、非连续密度梯度离心法(Ⅱ法)2种Percoll分离方法进行全面组合,共获得8种组合条件,分离小鼠肝脏淋巴细胞,用流式细胞术检测各类型细胞比例。结果:肝脏预处理方法中B法+C法获得淋巴细胞比例最高,A法获得淋巴细胞绝对数最高,B法获得的NKT细胞和NK细胞比例和细胞绝对数均最高;Ⅱ法获得的淋巴细胞、T细胞、NKT细胞和NK细胞比例和细胞绝对数均高于Ⅰ法。对于NKT细胞比例和NK细胞绝对数,肝脏预处理方法和Percoll浓度之间有交互作用(P0. 01和P0. 05),其他均无交互作用(0. 05)。结论:采用摘眼球放血结合体循环灌流法,结合Percoll非连续密度梯度法(33%+70%),可获得较高数量和比例的固有免疫细胞,有助于对肝脏固有免疫细胞如NKT细胞、NK细胞等的研究。Abstract: Objective: To compare and define the suitable range of the different Methods for isolation of murine hepatic lymphocyte，natural killer T cells( NKT cells) and natural killer cells( NK cells) . Methods: According to factorial design， four liver pretreatmentMethods including unbleeding method( A method) ， the eyeballs bleeding method( B method) ，body perfusion method( C method) ， the eyeball with body perfusion method( B method + C method) and two percoll isolation Methods including single concentration method( Ⅰ method) and continuous density gradient method( Ⅱ method) had been combined into 8 kinds of combination conditions， four bleeding Methods were used to prepare cell suspension respectively，and the single concentration method ( 33% Percoll) and the continuous density gradient method( 33% Percoll + 70% Percoll) were used to separation of mouse hepatic lymphocytes respectively，the hepatic innate immune cells were analyzed with two or three-colors immunofluorescent-labeling. Results: With the analysis of variance of factorial design， the highest proportion of lymphocytes was obtained by B method + C method， the highest absolute lymphocyte count was obtained by A method， the highest NKT and NK cell percentage and absolute number was obtained through B method; the ratios and the number of lymphocyte，T cell，NKT cell，NK cell obtained from B method were higher than those of A method. The ratio of NKT cell and NK cell absolute count， liver pretreatment method and percoll concentration had interactions each other( P ＜0. 01 and P＜0. 05)， the others were no interaction( P ＞0. 05) . Conclusions: High proportion of natural immune cells can be obtained by bleeding with perfusion combined with the continuous density gradient method( 33% + 70%)， it is helpful for research on NKT cells and NK cells.
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Keywords:
- cell separation /
- hepatic lymphocytes cell /
- isolation methods /
- percoll /
- factorial design
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