Hi everyone,
This is the first time I try to stimulate RAW 264.7 using LPS, then after that I will treat with drug and measure for the amount of TNF-alpha. I have some question:
1. How many of cells should I seed into 96 wells plate so 100uL of 100ng/ml of LPS can stimulate those cells?
2. After seeding the cells and incubating overnight for the cells to attack to the wells, do we need to discard the medium before putting LPS in media into the cell?
3. When should I put my drug (my drug is dissolved in media), and if I put drug into the well, so the concentrate of LPS and drug will change, so what should I do?
Thank you very much
Seed 1-2x10e5 cells/well of a 96 well plate. Leave them overnight so cells have time to attach and recover. The next day, remove the medium and add 100 uL LPS containing media (10-100 ng/mL). Then add 100 uL of drug containing media (at your desired concentration). If you are worried about the drug/LPS diluting upon addition to cells, make 2X LPS/drug solutions, so the final concentration in the well would be what you want (1X).
Can't tell you when to add your drug to the wells. It would depend on what your drug is and what you hope to achieve by adding it. Check the literature and see what others have done.
Dear Anh,
I am attaching a paper where we describe what we did with RAW cells exposed to LPS. I hope this can help. Good luch, Thierry
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