One of student is studying the methylation pattern of certain interleukins from the patients of rheumatoid arthritis. In start of process, he is using Histopaque-1077 based method to extract mononuclear cell layer. But even after following exact protocol (Image attached of protocol used), layers are not being formed? Can anyone guide to make it work?
Dear Azam Ali, the protocol in general looks fine to me - Particularly because it is provided by the manufacturer. Common problems with PBMC isolation are disturbing the gradient when not carefully handling it, centrifuging without break, and pipetting too much of the supernatant (= plasma) after the centrifugation step.
As an alternative, I can provide you with our protocol for PBMC isolation from RA patients using Ficoll Histopaque too. It is based on 50 ml Falcon tubes with a filter (available e.g., by Greiner bio-one). But of course, it also works with 50 ml Falcons without filter. This then only requires careful pipetting of the blood on top of the ficoll. The last to steps in our protocol are only required if you want to submit your cells to FACS analysis.
Please do not hesitate to contact me in case of further questions about it.
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