how purify cd4 t cell from PBMC,
does the cd4 t cell could be store in liquid nitrogen?
Dear Ming Lu,
For CD4+ T Cell sorting you can use FACS, if you don't have FACS still you can sort out by negative selection method using magnetic tagged antibodies against other cells except CD4+ cells which under strong magnetic field gets attached towards the magnet and the solution retains your cells of interest. I have use BD magnetic cell sorting kit for CD4+, CD8+ and NK cells from murine splenocytes it worked well for us, they also provides magnet for sorting.
Regarding freezing the stored CD4+ cells, I have not tried it since its a primary cells we use to screen the drugs immediately once we sort them.
Note: you have to sort macrophages for co-culturing with CD4+ since they are the essential for CD4+ cell survival
Hi,
I agree with the others, you can isolate CD4 T cells by positive or negative selection by using magnetic beads. I used the Miltenyi Biotec in the past and they work very well, it is very easy protocol and then you can use these cells for culture later or face staining, or you can isolate by sorting them too.
Regarding the cells storage, what I did before is storing the PBMCs in liquide Nitrogene, and once I needed I unfroze them and isolate the needed cells like CD4 T cells with no problems, you may loose some but if you store a good number of PBMCs you will still have enough after (of course depending of what you want to do).
Good luck :)
We have used Miltenyi Biotec CD4+ T cell enrichment kit (column-based), Stemcell Technologies CD4+ T cell enrichment kit (magnet) and Affymetrix Magnisort CD4+ T cell enrichment kit (magnet) and they all work well. Magnisort is the cheapest of them. The advantage to use an enrichment kit is that the CD4+ T cells remain untouched until you stimulate them and you get between 95 and 98% purity. Watch out if you use CD4 microbeads as they can capture CD4 T cells AND monocytes. Quiescent CD4+ T cells can be stored in liquid nitrogen in 10% DMSO and 90% FBS. Wait at least 16h after you have thawed them before stimulation.
To induce proliferation, you can use anti-CD3 with or without anti-CD28 or phytohemagglutinin (PHA-L; 1 ug/ml). It is important to add IL-2 (10 to 50 units/ml) at least the day after the stimulation. We coat cell culture plates with anti-CD3 (5 ug/ml) and anti-CD28 (2 ug/ml) in PBS overnight at 4C and then we remove the PBS and stimulate the cells in the coated wells with 30 U/ml of rhIL-2. The number of live cells will decrease in the first two days but after 3 days, the cells will have recovered entirely, will continue to proliferate and can be infected with HIV.
Good luck,
Michel.
Monocytes represent between 5 and 10% of PBMCs but after CD4 microbeads separation, they will represent between 15 and 30% of your CD4+ cell population. However, the monocytes will rapidly adhere to plastic so you could plate the CD4+ cell population (containing monocytes and T cells) between 2h and 16h and only take the cells that are in suspension. You would get a purer CD4+ T cell population this way.
Another thing to keep in mind is the possibility that CD4 triggering by the beads will affect their differentiation.
Best of luck,
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