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sanger09新虫 (小有名气)
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[求助]
求细胞粘附实验的具体步骤
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各位大神，我本科不是学生物的，跨专业考过来的，现在要做个细胞粘附实验。看了好久，不知道怎么做，所以来小木虫发帖询问。 各位能够告诉我，细胞粘附实验的具体步骤是怎么样的？ 越详细越好。 |
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8楼2013-03-16 16:42:31
sanger09
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2楼2012-06-03 21:21:09
peopole
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3楼2012-06-04 08:56:53
xuzhengxin405
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【答案】应助回帖
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zhaohq1209: 金币+3, 有帮助~学习了~ 2012-06-05 08:40:35
感谢参与，应助指数 +1
zhaohq1209: 金币+3, 有帮助~学习了~ 2012-06-05 08:40:35
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给你两个protocols，我当时就是参照这些资料做的 Adherence assay CHOK1 cells were grown in 24-well tissue culture plates to approximately 80% confluence. Cells were transfected with either pCMV5 Nrx1β-mCherry or pCMV5 FLAG-Nrx1β-mCherry using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and grown for 36 hours to allow for protein expression. For attachment assays, bacteria were washed 2 times in PBS, resuspended in CD CHO media and incubated for 1 h with CHOK1 cells at an MOI of 10. To remove unattached bacteria, cells were washed 4 times with PBS. To determine the number of attached bacteria, CHOK1 cells were lysed with sterile water and serial dilutions plated on the appropriate media. The number of attached bacteria was reported as a percent of total bacteria at the end of the incubation period. Each experiment was performed in triplicate wells and repeated three times. Statistical analysis was performed using the Student's t-test. Adhesion assays A549 cells (human alveolar type II pneumocytes; ATCC CRL-185), were grown to 90% confluence on 24-well plates (~106 cells/well). Prior to use, cells were washed with cell F12 media to remove serum. For competitive inhibition binding assays, A549 cells were incubated with 1?M of either rBR, rBR.C, a synthesized peptide corresponding to AA 122-167, or BSA for 1 HR. Following incubation, cells were exposed to media that contained 107 cfu/mL of bacteria and incubated for 1 h at 37°C in 5% CO2. Nonadhering bacteria were removed by washing the cells 3 times with T-PBS and the number of adhering bacteria was determined by lysis of the monolayer with 0.1% Triton X-100 and plating wells per experiment. |
4楼2012-06-04 09:35:29