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PBMCs culture

Stimulation with PMA PBMCs stimulation

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#1 Divya

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Posted 30 September 2012 - 05:57 AM

Hi Folks,
I have put culture in 24 wells plate, used PMA (15, 20, 25 ng/ml), Ionomycin (1.5, 1, 0.5 microgram/ml) respectively and monensin (2.5 microliters, 2mM) each in 3 wells and one well with unstimulated cells (>1 million cells/well), have used complete media (filtered RPMI with 10% FCS) at 37 degree C for 6 hours but >90% of the cells (even unstimulated) died. What could be the possible reasons of the cell death? How should I standardize the PBMCs culture? Which protein transport inhibitor is better Monensin or Brefeldin A if I want to look Th1 (IFN-g), Th2 (IL-13) and Th17 (IL-17) cytokines.

I would be thankful if anybody suggest me protocol/article to go through. What other precautions should I need to concern during culture procedure?
Thanx
XYZ

#2 Divya

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Posted 30 September 2012 - 06:25 AM

Hi Friends,
I wanted to standardize the staining for Intracellular cytokines like IFN-g (Th1), IL-13 (Th2) and IL-17 (Th17), for that I have put culture of human PBMCs in 24 wells plate, used PMA (15, 20, 25 ng/ml), Ionomycin (1.5, 1, 0.5 microgram/ml) respectively and monensin (2.5 microliters, 2mM) each in 3 wells and one well with unstimulated cells (>1 million cells/well), have used complete media (filtered RPMI with 10% FCS) at 37 degree C for 6 hours but >90% of the cells (even unstimulated) died after having counted via trypan blue staining on hemocytometer. What could be the possible reasons of the cell death? How should I standardize the PBMCs culture? Which protein transport inhibitor is better Monensin or Brefeldin A. I have made fresh complete media from incomplete RPMI, which i already prepared 2 months before and stored at + 4 degree C n was red in color.
I would be thankful if anybody suggest me protocol/article to go through. What other precautions should I need to concern during culture procedure?
Thanx in advnc




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