Hi.i wanna culture lymphocyte cell from whole blood.any body have any recommend?does it need any special care or just 10% fbs,antibiotic,dmem?
If you would like to make passaged culture of not activated white blood cells, I can't help you - I didn't succeed. A common activating agent for WBC is PHA, especially for T-cells. You usually get high apoptotic levels on 1-5 days, but in 4-5 you can see a subpopulation of Ki67-positive cd3-positive cells (proliferating T-cells of normal morphology and immunophenotype). I culture WBC for 1-2 days (survival culture) using 15% FCS and RPMI. There are many rumours on the amount of serum and media type, including serum-free hybridoma-type media, but for me there is no big difference. Next, I prefer not to use antibiotics since they can harm cell respiration or cytoskeleton. Finally, B-cell culture is a more difficult thing since B-cell are much more delicate than T-cells.
so i need to stimulate them.i read that i can do stimulation with either PHA or LPA.i knew that they are very sensitive but i didn't expect so much.and i would like to know the procedure after taking blood from donor.furthermore i would like to ask if the cells are ok with out antibiotics?do you think i would have problem with 10%fbs instead of 15%fcs?
thank you
what i know blood have many cells like WBC,RBS,platelates. to get pure population of lymphocyte u load the blood on percoll gradient. keep it for 15 min at 10000 RPM in centrifuge.This leads to formation of 4 bands: a layer containing dead material (if present) which did not enter the gradient; a layer near the bottom of the tube containing granulocytes and red cells, and two other bands in between. The upper one is enriched for monocytes and the lower one for lymphocytes . collect the cells from lower layer. seed into in vitro culture with medium dmem+15 % FBS+1 % antiiotic.later u can add GMCSF (after 5th passage)as growth factor.
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