细胞药筛-CCK-8法.docx

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1、实验方案CCK-8法评估化合物对细胞系的细胞增殖影响实验一实验目的：本实验的研究目的是用CCK-8法评估药物对细胞系的细胞增殖影响。通过检测在不同药物 浓度处理后的细胞抑制率及细胞活力，计算50%抑制浓度。二实验设计：在4株细胞中测定2个化合物，每个化合物9个浓度，3个复孔，72小时后用CCK-8法检 测细胞活力，并计算IC50。三实验材料：L 实验细胞：NCI-H23、Mia-paca2 NCI-H358, SW837 (细胞将置于 37、5%C02 和 95% 湿度条件下培养。)2 .实验试剂与耗材：细胞培养常规培养液和耗材胎牛血清 FBS (GiBco, Cat# 100099-1410

2、Cell Counting Kit-8 (碧云天，Cat# C0039)96孔透明平底细胞培养板(Corning, Cat# 3599)3 .实验药物：编号名称浓度(mM)分子量溶剂储存1AMG51010DMSO-20C02Adagrasib10DMSO-20C四实验步骤：(-)细胞培养：细胞复苏并培养于各自的培养液中。(至少传代一次以上，细胞状态)(二)细胞铺板密度选择1 细胞系：Mia-paca2 (DMEM+10%FBS+2. 5%HS)NCI-H358 (RPMI1640+10%FBS)NCI-H23 (RPMI1640+10%FBS) SW8372优化细胞密度：2. 1铺板：96孔板

3、中铺入三种细胞(最终密度为16最Ocells/well, 8000cells/well, 4000cells/well, 2000cells/well, lOOOcells/well, 500cells/well) 200ul/孔Mia-paca2NCI-H358NCI-H23SW837160001600016000160001600016000160001600080008000800080008000800080008000400040004000400040004000400040002000200020002000200020002000200010001000100010001000

4、1000100010005005005005005005005005002.2 其余无细胞的孔使用PBS补充。2.3 显微镜下观察细胞72h后，细胞融合度为70-80%的细胞密度为好。二细胞药物筛选：1 ,根据每株细胞所选好的密度铺板（90ul/孔），其余无细胞的孔使用PBS补充。如下图:Row123456789101112ABCellCellCellCellCellCellCellCellCellCell空白AMG510CCellCellCellCellCellCellCellCellCellCell空白DCellCellCellCellCellCellCellCellCellCell空白E

5、CellCellCellCellCellCellCellCellCellCell空白AdagrasibFCellCellCellCellCellCellCellCellCellCell空白GCellCellCellCellCellCellCellCellCellCell空白H2 .细胞贴壁24h后，选择合适的药物浓度加入，如下图:C9C8C7C6C5C4C3C2ClCO空白AMG5102512.56.251.56250.3906250.0976560.0244140.0061040.0015260空白2512.56.251.56250.3906250.0976560.0244140.00610

6、40.0015260空白2512.56.251.56250.3906250.0976560.0244140.0061040.0015260空白Adagrasib103.1601.000.3160.100.03160.010.003160.0010空白03.1601.000.3160.100.03160.010.003160.0010空白03.1601.000.3160.100.03160.010.003160.0010空白3加药72h后进行CCK-8检测。参考CCK-8说明书。附：药物配制AMG-510母液C9C8C7C6C5C4C3C2Cl溶剂DMSO培养液培养液培养液培养液培养液培养液培养

7、液培养液培养液浓度10mM250nm125 口 m62.5小15.625|dM3.906|jM0.977W0.244W0.061kiM0.015nM体积80.180|jl12031203120Hl1203120312031603稀释比lin401 in 21 in 21 in41 in41 in4lin41 in41 in4孔内体积43156Hl- te80Hl1203120Hl120pil120Hl120Hl取出体积801jL80日”40tiL0 4加 L f4的L4M夕4血”4ML ：110倍稀释（10pl药物溶液+ 901dl培养液）C9C8C7C6C5C4C3C2Cl溶剂培养液培养液培

8、养液培养液培养液培养液培养液培养液培养液浓度(lx)25川1.25iM6.25川1.5625|jM0.3906 口 M0.0977nM0.0244W0.0061|jM0.0015|jM体积100|jl1003100|jl100|jI100|jl100|jl10031001100口1稀释比1 in 101 in 101 in 101 in 101 in 101 in 101 in 101 in 101 in 10Adagrasib母液C9C8C7C6C5C4C3C2Cl溶剂DMSODMSODMSODMSODMSODMSODMSODMSODMSODMSO浓度（400x）10mM4mM1.27mM4

9、00HM127(jM40HM12.7|jM4HM1.27W400nM体积120|dl21闻121.6W21.6321.6321.6321.6kil21.6131.6小稀释比1 in 2.51 in3.161 in3.161 in3.161 in3.161 in3.161 in3.161 in3.161 in3.16体积2W-J181dlA21.6321.6口121.6|jl21.634 +21.6W21.63夕*2L6W 另文21.6|dl4体积310|jLr 10|jLJ 10|jLf 1O|JLr 10|jLlOkiL10|jL10|jL/40倍稀释（5|jl药物溶液+ 195Hl培养液）

10、C9C8C7C6C5C4C3C2Cl溶剂培养液培养液培养液培养液培养液培养液培养液培养液培养液浓度 (10x)100nM31.6|jM10|jM3.16|jM1|jM316nMlOOnM31.6nMlOnM体积42004200Hl200Hl20032003200Hl200|jl200八12003稀释比1 in 401 in 401 in 401 in 401 in 401 in 401 in 401 in 401 in 4010倍稀释（10口1药物溶液+ 90kli培养液）C9C8C7C6C5C4C3C2Cl溶剂培养液培养液培养液培养液培养液培养欣培养欣培养欣培养液浓度(lx)10|jM3.1

11、6小1|jM316nMlOOnM31.6nMlOnM3.16nMInM体积5lOOkil1003lOOnllOOpil1001lOOpI10031003100|jl稀释比1 in 101 in 101 in 101 in 101 in 101 in 101 in 101 in 101 in 10注释： 体积1：梯度稀释后所得DMSO溶液体积体积2: DMSO溶剂体积体积3: 梯度稀释时转移到下一浓度的DMSO溶液体积体积4: 培养液稀释后所得体积体积5:96孔板中的终体积 细胞存活率=（实验孔一空白孔）/ （对照孔一空白孔）X 100% 细胞抑制率=（对照孔一实验孔）/ （对照孔一空白孔）X 100% 实验孔（含有细胞的培养基、CCK-8、待测物质） 对照孔（含有细胞的培养基、CCK-8、没有待测物质） 空白孔（不含细胞和待测物质的培养基、CCK-8）注意事项：如果待测物质有氧化还原性，可在加CCK-8之前更换新鲜的培养基，去掉待测 物质的影响。