应用CCK8法检测鸡淋巴细胞活性的检测最佳条件研究

发布时间：2018-03-30 21:24

本文选题：CCK-法　切入点：鸡　出处：《黑龙江畜牧兽医》2017年13期

【摘要】：为了研究应用CCK-8法检测鸡淋巴细胞增殖的体外培养最佳条件,试验设置了不同种板细胞数、血清浓度、培养时间、孵育时间和检测波长等条件对其进行比较研究。结果表明:1 750 r/min转速的改良鸡外周血淋巴细胞分离方法效果最佳;在体外增殖培养试验中,CCK-8加样4 h后在450 nm波长处检测OD值最理想,培养时间以44 h为最佳;在细胞数量一致的情况下,胎牛血清浓度为5%或者为10%差异不显著(P0.05)。当细胞悬液浓度为1×10~6个/m L时,加入100,50,25,12.5μL不同细胞数,4组间比较均差异显著(P0.05),并且随着细胞悬液浓度的增加OD值增加;最佳铺板细胞的细胞悬液浓度为2.0×10~6~3.0×10~6个/m L,取50~100μL为宜,即最佳细胞铺板数量控制在每孔1.5×10~5~2.0×10~5个/100μL。说明应用CCK-8法检测鸡外周血淋巴细胞活性的最佳条件除种板细胞数不同外,在检测波长、血清浓度、加样后孵化时间等方面与其他动物试验差别不大。
[Abstract]:In order to study the optimal conditions for the in vitro culture of chicken lymphocyte proliferation by CCK-8 assay, the number of different kinds of plate cells, the concentration of serum and the culture time were set up. The incubation time and detection wavelength were compared. The results showed that the improved method of separating peripheral blood lymphocytes at 1: 1 750 r/min speed was the best. In vitro proliferation culture test, the best OD value was detected at 450 nm wavelength after adding CCK-8 sample for 4 h, and the best culture time was 44 h, when the number of cells was the same, There was no significant difference in fetal bovine serum concentration of 5% or 10%. When the cell suspension concentration was 1 脳 10 ~ 6 / mL, there were significant differences among the four groups (P 0.05) when the cell suspension concentration was 1 脳 10 ~ 6 / mL, and the OD value increased with the increase of cell suspension concentration. The optimal cell suspension concentration was 2.0 脳 10 ~ (6) ~ 3.0 脳 10 ~ (-6) / mL, and 50 ~ 100 渭 L was chosen as the optimum cell layer, that is, the best cell layer number was 1.5 脳 10 ~ 5 ~ 2.0 脳 10 ~ 5 / 100 渭 L per well. The best condition for detecting the lymphocyte activity of chicken peripheral blood by CCK-8 method was different from the number of seed plate cells. The detection wavelength, serum concentration, incubation time after adding samples were not different from other animal tests.
【作者单位】：温州科技职业学院;浙江省农业科学院畜牧兽医研究所;浙江省亚热带作物研究所;
【基金】：2015年浙江省新苗人才计划项目(2015R458001) 浙江省教育厅高等学校访问工程师校企合作项目(FG2014197) 浙江省重大优先项目(2008C12044-3)
【分类号】：S831

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