Dear all,
I´m working with human monocyte-derived macrophages and would like to analyse them by flow cytometry. After monocyte isolation I differentiate them with 100 ng/ml M-CSF for 6 days and tried numerous ways to detach them from the dish for analysis.
I tired without success:
- incubation with PBS+2mM EDTA --> no detachment
- incubation on ice for up to 1h in PBS+2mM EDTA --> no detachment
- scraping cells with cell scraper --> all cells detached but >50% dead cells visible during flow cytometry
- Macrophage Detachment Solution from PromoCell on ice for up to 1h --> moderate detachment
- Accutase treatment at 37°C for up to 1h --> moderate detachment
- TrypLE treatment at 37°C for up to 1h --> moderate detachment
Donor variability results in varying cell recovery for all detachment procedures between 20-50%.
Incubation on ice using UpCell plates from NUNC is working very well but these plate are quite expensive and one-time use.
Can anyone recommend a procedure that works reliably?
Thank you!
Dear Bianca,
I have done some MDM assays a while ago, currently not working with them anymore, however, I had very good experience with the PromoCell solution.
We had a superb recovery and very little dead cells (well over 90% live cells).
Do you wash the cells before you add the PromoCell solution (if I remember correctly removing FBS/serum was important)? I was basically following the manufacture's instructions, however, I think a important factor (and you have also mentioned that) are the plates or more precise, the material/treatment. I was wondering, do they recommend to incubate the cells with the cell detachment solution for that long (1h)?
We were using nunclon t25 flasks, which are a bit more expensive, however, it was very much worth it.
Best,
Johannes
Dear Blanca
I don't know how expensive are Corning Ultralow-Atachment plates compared to UpCell plates but 30min incubation on ice with PBS + 2mM EDTA works perfect for us to detach monocyte derived macrophages by washing with a tranfer pipet. (Viability always above 85-90%)
Before these plates, we used autoclavable teflon flasks with same treatment and they also worked but sometimes it was very difficult see cells through these flasks.
Hope it helps
Try to use untreated plates (sterile), for example, those Petri dishes used per bacteria growth. I found most of the time cheapest plates work better.
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