I'm looking to isolate monocytes from thawed PBMCs, by negative selection using MACS. I usually rest overnight, but I'm worried that the monocytes will adhere to the culture plate. Does anyone have any advice on how to isolate monocytes from frozen PBMCs? Thanks.
Monocytes need about 30min at approx. 36°C and they will adhere to the surface (especially to plates from e.g. COSTAR). When I work with monocytes or PBMCs containing monocytes and I want to make sure that they will not adhere, I put them on ice (but not too long that will activate the cells). In case I have already had the cells for longer than 10min at RT and I want to remove the monocytes (you will see them attaching when you put the cells (monocytes or PBMC) under the microscope. The round monocytes become the shape of an fried egg)), I deattach them by: removing the medium and adding instead EDTA-Buffer (1mM EDTA, 1xPBS in H2O, pH 7.4) to the cells until they are well covered, incubating them on Ice in the fridge (4°C) for 10min. I Harvest them by washing the cells from the bottom of the well with the buffer, meaning not scratching but pipett the buffer against the bottom with a bit "push". You can check the result by microscope. Collect the cells (on ice) and sustitude the buffer with an appropriate medium (cold) or buffer (cold) after centrifuging (300xg, 7min, 4°C). Hope that helps
We keep monocytes in snap-cap polypropylene tubes for up to 24h at 37C. They adhere to that type of plastic much less than to polystyrene.
Article HIV-1-Triggered Release of Type I IFN by Plasmacytoid Dendri...
- Badges
- Science topic
- Similar topics
- Geoscience
- Cartography
- Mapping