I work with DT2 patients and I need to isolate monocytes from lymphocytes in my experiment. Is there any simple efficient and non-expensive method . Right now im trying this:
Extraction of PBMC with Ficoll
PBMC in flask with DMEM medium serum free.
2X106 cel/mL, total volume 10 mL
Culture: 3 hr, 5 % CO2,
Wash with DMEM serum free x3
Then wash with PBS/0.02% EDTA at 4 ºC for 10 minutes.
when i count it i see some limphos and viability with tripan show me some dead monos
somebody help me, please.
what Im doing wrong?
I prefer adherence isolation
I would prefer you use the monocyte isolation kit II from miltenyi. you can use the RPMI media and culture 2-3 days. Its pretty easy
Did you try EDTA at room tempereature? It could take longer (even 20 mins) but the viability can improve. If not try scraping. I agree with Sepiso if you want to have an highly pure population. For functional assays like migration or penetration is not needed though.
Thank you so much!, but I chose plastic adherence method and discard MACS and flow isolation. Someone told me that monocytes isolation should done with cold temperatures (4ºc) to increase viability. what do you know about that?
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