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anyone has experience in adhesion assay?

icam lfa adhesion t cell

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#1 gyma

gyma

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Posted 20 May 2013 - 11:19 PM

hello everyone I had a problem in adhesion assay.

I was testing the effect of a drug on T-cell adhesion. I coated recombinant ICAM1 in 96-well plate overnight at 4C and blocked it with 0.5%BSA for 1 h at 37C the next day. Then I seeded 5X10^4/well of T-cells (which have been confirmed to express LFA1, the ligand) and incubated the plate at 37C for 2 hrs. Here comes the problem. Most references just say that non-adhered cells are washed away. But how? I added PBS and then removed it immediately. To make sure that remaining cells were really adhering to ICAM1, I washed 3 times. And finally only very few cells remained. How should I wash the wells? Anyone has experience? Please tell me your advice, thank you.





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