hi.
i have a peculiar problem while culturing PBMC from human as well as mice splenocytes. for the picture part, I isolated T-cells using Ficoll-paque gradient (sigma), and then depleted monocytes by overnight incubation in T75 flask in RPMI (10% FBS. 1% P/S, and 2mM glutamine.
Each Day I have increased in left population. On Day 1 the left population marked by arrows is 13% of total cells and right large population is 61% but on Day 7, the right part is 38% and the left part is 31%. The right one are all alive or atleast 95% (PI negative). while the left population is 50% alive.
Moreover, I have seen different donors behave differently e.g. the above situation is for donor 1, while another donor had only 17% at Day 7 of good population and 53% of the left population. Even on Day 5 the condition of the cells are not good.
I am optimizing protocol for T-cell proliferation assay and MLR. but this shift in population is quite annoying me. Do anyone have suggestion what am I missing here?
Another important thing is when i added concanavalin A at higher concentration, majority of cells shifted to left population.
I didnt have IL-2 but I will repeat the experiment next week with IL-2. Still if anyone has any suggestion i will be thankful.Do I need 2-ME to culture human PBMCs?
These cells ( as of my experience) are high SSc B lymphocytes. I have not found any such data from proliferation assay, but in whole blood phenotyping I found in few patients high SSc lymphocytes. If you do CD19 staining for these, you should get maximum percentage of B cells in it.
Adding IL2 in culture is good for health of cells (during any stimulation).
BTW, T cells proliferation assay protocol is of 72 he, why would you go for 7 days culture??
Do you replenish Media after 2 days in culture? If you don't do so, your cells may undergo degeneration.
What stimulant do you use? It Decide the fate of cells.
Hi Jitenda,
Thanks for detailed response.
I am not sure about B-cells as these cells come from the low SSC cells and increase day by day, but i will try to do phenotyping to see which cells are there.
7-days is for MLR, i use lower concentration of ConcanavalinA, so i go for 5 days Proliferation assay.
I dont replenish, so that might be the issue (but again its happening on Day 2 also, so that might not be the reason, still I will try).
For IL-2 I was afraid it will cause stimulation of T-regs and may affect MLR, therefore i didnt want to add.
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