Mohamed,Did you find way to make the cells to grow?Thanks

TT
Thao Tran
Feb 11, 2015
protocol Protocol: In vitro Culture of Human PBMCs
Mohamed,

Did you find way to make the cells to grow?

Thanks
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Thao Tran Answered Feb 20, 2015

Texas A&M

I counted by hemocytometer. Over the time, the cell population decreased. I am using LPS from sigma and PHA ftom Gibco.
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Balachandran Ravindran Author Answered Feb 19, 2015

Infectious Disease Biology, Institute of Life Sciences, India

Can you clarify when you say 'nothing worked?!' I guess there was no proliferation of cells PHA or LPS. How did you score proliferation? How sure you are about the quality of PHA or LPS? Adding 10 times more PHA or LPS could actually be toxic to cells.
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Thao Tran Answered Feb 11, 2015

Texas A&M

I activated PBMC with either PHA 5 ug/ml or LPS 1 ug/ml. I used medium IMDM with 20% bovine serum. After 4 days, nothing worked, the cell population decreased! Now I increase the PHA and LPS up to 10 times concentration. I hope it will woek (after the first day, I checked but it still has not been working well). How long is the wait? Any one could provide me suggestions?
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Balachandran Ravindran Author Answered Feb 11, 2015

Infectious Disease Biology, Institute of Life Sciences, India

Cells in PBMC constituting Monocytes and Lymphocytes are terminally differentiated cells normally. Monocytes do not divide and can only be differentiated to macrophages by adding cytokines into culture. Lymphocytes also will not divide unless activated by a variety of means. Under normal culture conditions cells will die over time.
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