Hello all,
I am currently performing a transwell assay with primary NK cells. I have tested this system under several conditions, including the addition of HIFBS, varying incubation times (1 to 2 hours), and different cell numbers. However, the results have not been consistent, particularly the chemoattractive effect of CXCL10 (50 nM), which is not as pronounced as reported in other papers. Could you provide some advice on how to improve this assay?
Thanks in advance!
Here are some general tips for consistent cell culture experiments
1. passage and confluency of cells prior to seeding for assay should be similar, especially for primary cells. I'm not familiar with NK cells but primary cells can change a lot from one passage to the next. keeping the passaging and state of the cells similar prior to seeding in the transwells should help minimize if cells were overgrown and might go into G0.
2. enough technical replicates (wells) per condition. I would use at least 4 so if there is a wonky one then it can be excluded and there are still 3. then the average of those 3 wells is one bio replicate.
3. aliquoting the stimulant chemokine - freeze/thaw cycles of this will affect its activity so aliquot it early, don't just use one tube over and over. also the source company for this protein, might vary from one to another.
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