I'm trying to cultivate PBMC (fresh or frozen) for 7 days and I don't see monocytes (CD14+) anymore at the end of the culture.
here is my protocol so far:
PBMC fresh: Ficoll and retrieve of the cellular ring.
or PBMC frozen
culture in 96 well plae U shape at 2x10^5 PBMC per 100uL in
DMEM
+ fetal calf serum
+ PS
+ glutamin
+ sodium pyruvate
+ sodium bicarbonate
+ non-essentiel amino acids
I need to stimulate them with a non toxic compound during the first 24 hours then do a wash out and let them rest for 7 days.
My compound is supposed to modify my monocyte activities but I can see them anymore.
have you any advice? Why am I losing the monocytes?
thanks in advance
Hello
Why don't you culture PBMC in RPMI +10%FBS. Low level of activation will be seen with time.
You can add PHA or LPS at 1-5ug/ml to induce proliferation and keep them going for 7 days.
Best Wishes.
Dear Alexis!
You look also at following protocol:
https://blog.quartzy.com/2017/05/30/peripheral-blood-mononuclear-cells-pbmc-isolation-preservation-culture
PBMC culture
PBMCs can be cultured for 5-7 days in 24- or 96-well plates, using supplemented RPMI-1640 medium, and incubated at 37°C in a humidified, 5% CO2 atmosphere. PBMCs do not readily proliferate without stimulus and should be plated at a density of 0.5-1 x 106 cells/mL in a total volume of 0.5-1 mL in a 24-well plate, and 200 µL in a 96-well plate. Because PBMCs comprise both lymphocytes and monocytes, some cells will adhere to the plate (monocytes/macrophages) while others may be in suspension (lymphocytes). This must be taken into account when culturing—for example, round- or V-bottom plates may be more appropriate if cells need to be pelleted by centrifugation. To induce proliferation, phytohemagglutinin (PHA) or lipopolysaccharide (LPS) can be added to the culture medium at a concentration of 1-5 µg/mL.
Dear Alexis, I agree with the previous comments.
Monocytes prefer RPMI medium and culture in high density eg 3x105 cells in 96 well plate in 200ul medium. Without stimulation they do not last more than 3 days in culture.
Hello Alexis, I would expect there to be between 3-15% monocytes in my fresh PBMC preparations.
The number and their function decline rapidly with storage time. Fresh PBMC monocytes will adhere rapidly to polystyrene(20 minutes) though to harvest, I check numbers using a flow cytometer(CD14), seed, as required, incubate for 2-4 hours, before removing the lymphocytes by simple washing the wells and culturing them on(->90%+ CD14). The lymphocytes are them added at specific ratios depending on the initial seeding numbers for the monocytes.
They may be lost after 7 days culture on plastic, due to activation, apoptosis, modulation or death but if you want to check they are there at the start, at same numbers under all starting conditions, you could try the two steps it takes little additional time.
Best wishes
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