Hello,
I'm trying to get 100% of human CD4 T cells to proliferate. My current workflow is to isolate the CD4 cells from PBMCs using a CD4 negative isolation kit (Miltenyi). I then culture the cells at 3,1,0.3x10^5 in a flat-bottom well (coated overnight with 9-1ug/mL anti-CD3) with 2ug/mL soluble anti-CD28 in complete RPMI. I'm using CTV-dilution as a read-out of proliferation. I can't seem to get more than 87% of the cells to dilute CTV, which correlates to 50% of my cells according to FlowJo's proliferation analysis software. Is there any way that I can get 100% of my cells to proliferate? Thanks.
I don't think you will have anyone say thta it is possible to get 100% of cells to proliferate. The reason being... there are other cells that express CD4. And the negative selection will always have more contimination than positive selection. And on top of this, ALL in vitro cultures, and certainly ALL cultures that uses any type of stimulants will cause some cells to just die. Not all cells are at the same stage.
It is normal as your CD4 negative isolation is not efficient at 100% so you have some contamination with other cell types. If you are sure that the cells in cell culture are CD4 , I will recommand to add IL-2 with CD3 and CD28 beads. Good luck
I don't think you will have anyone say thta it is possible to get 100% of cells to proliferate. The reason being... there are other cells that express CD4. And the negative selection will always have more contimination than positive selection. And on top of this, ALL in vitro cultures, and certainly ALL cultures that uses any type of stimulants will cause some cells to just die. Not all cells are at the same stage.
It is always difficult to get the proliferation of all the CD4+ T cells, but maybe it is a matter of time: How long your cells stay in culture?. Another possibility is that maybe you are using a too high dye concentration so there is some cell toxicity; maybe you colud try to reduce the amount of CTV per cell....
Good luck
Fran
Hi Christopher,
It is possible to get close to 100% proliferation, provided you optimize the culture conditions. A few suggestions that work well in my hands:
1. Use round bottom instead of flat bottom plates for antibody coating (assuming you are using 96 well format).
2. Coat antibodies in 0.1 M sodium bicarbonate buffer pH 9.2 instead of PBS.
3. Coat antibodies at 2.5 ug/mL concentration, and also coat the aCD28 (soluble stimulation isn't optimal when there are no Fc receptor-expressing cells in your culture).
4. Use appropriate antibody clones for T cell activation (e.g. aCD3 UCHT1 and aCD28 CD28.2 work well).
5. Add IL-2, but don't add IL-7 to the culture. The latter will keep your unstimulated cells alive, and if your goal is 100% proliferation you rather want those to die by neglect.
6. Give sufficient time, at least 4 days before doing the analysis.
Good luck!
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