Hello.
I am a student in master cycle, I would like to study the activation of the inflammasome in vitro in human monocytes, we do not have a monocyte isolation kit so I use the classical technique of isolation by ficolle and the selection of monocytes by adhesion. as a culture medium I use RPMI 1640 with 10% FBS and 1% L-glutamine and 1% penicillin / streptomycin. is that correct? and what precautions do I need to follow to have good cell performance and monocytes in good condition?
The media looks fine. (I never used antibiotics...but that's a personal preference.)
The only other thing I would add is you to make sure you do a verification of your monocyte population by flow.
I isolate monocytes by 1st step by Ficolle density gradient and then in the 2nd step Percoll density gradient. Finally, monocytes population is confirmed by flow cytometry. Base media is RPMI.
The ideas above are right but ensure you use appropriate RPM during the centrifugal process.
The ideas above are right but ensure you use appropriate RPM during the centrifugal process.
Thank you for your answers Emmanuel Ifeanyi Obeagu Hasan Al Faruque Shen-An Hwang
In my experience, you will definitely have to add some sort of "enrichment" step if you want viable monocytes. Simple adhesion doesn't work; you can add an additional Percoll step after Ficoll.
In the very first step after isolation of PBMCs, I would never use 10% FBS. Instead, if you are isolating human monocytes, you would better use 0.5-1% human serum which is provided by Sigma or other manufacturers. The idea behind using low serum, is that the high concentrations of FBS, have negative effects on your purity and lymphocytes will not be washed away properly. Thus, after almost 3 hours of incubation, the purity of attached cells would be more reliable. Then, you can wash the remaining cells which are mostly lymphocytes and detach your cells using ice-cold EDTA 5mM.
Best of luck
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