I stimulated RAW264.7 using LPS(100ng/ml) + IFNgamma(20ng/ml) for 24h,then using WB to detect CD80,CD86 and inos,inos is obviously increased but CD80 and CD86 are decreased.What is the reason for this? Thank you for your guidance!
In addition, during the induction process, vacuoles were found in RAW264.7 macrophages, why is this?
Hi, Joyce.
Thank you for your response. It is true, as you say, that inos increases significantly and reproducibly. However, I did not get a steady increase in CD86 protein levels and I have not performed flow cytometry yet. cd86 is a membrane protein and I am not sure if it makes sense to detect it by WB. Also is there a time window for LPS+IFNgamma to induce changes in CD86? I have set time points of 0,2,4,8,12,24h and have not got reasonable results, which confuses me. Looking forward to your reply if you get an answer.
Thanks,
KunZhang
Deborah Joyce
Kun ZhangHi Kun Zhang,
l had detected the expression of CD80 and CD86 in RAW264.7 stimulated by single 1000 ng/mL LPS for 12/24 hours by flow cytometry, indicating that the percentage or MFI (similar to the level of WB) were both elevated. Maybe you could try to increase the concentration of LPS from 100 ng/mL to 1000 ng/mL.
And other details should be considered:
- The RAW264.7 cells were easy to polarize to M1 phenotype spontaneously. Please use another one cell line if you find the cells aren‘t nummular (M0 phenotype).
- Don't filtrate, which will inactivate LPS.
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