i am coculturing t cells with other cell types and after co culture i want to isolate only t cells and eliminate other cells which this t cell is interacting so which method can i follow let it be magnetic sorting etc which one will give good results if any one has did this please let me know the procedure.and if i do this the interaction between the two cells will be broken and will be getting pure cell population?
It would help if you know what the other cell types might be. If it's possible you can treat the other cell types with mitomycin D or gamma irradiation and they will not proliferate. In theory it should be possible to get a rather T-cell population by using anti-CD3 (or CD4 , CD8) magnetic beads (we used to use dynal or miltenyi). You can stain for CD3 on the input for the magnetic bead separation and column fraction, then run them on the flow cytometer. I have done this for a mixed-lymphocyte reaction before, and you can still retrieve T-cells after that. The interaction would be broken, but this can definitely be checked on the flow cytometer.
Hello Gen Lin,
I have been using Tcells and Adipocyte coculture and here i want to isolate t cells from this culture and what i want to tell you is this adipocyte is a adherent culture to that we will add t cells and will be looking at the interactions and here if we want to study t cells from this culture we need to isolate pure T cells from this co culture and what we have found is these t cells will be interacting with adipocytes and will be in contact so what can be done to disrupt the attachment and etc i want to know. here the adipocytes were differentiated from a preadipocyte culture in vitro and these adipocytes at this stage will be growth arrested and will not proliferate so from what step we can start this isolation.Add your answer
I have purified naive T cells from PBMCs with miltenyi magnetic columns getting a yield of over 95%. They have great kits.
Ideally, check in the bibliography how to dissociate the two supopulations you are co-culturing.
You could use magnetic beads or a similar approach to sort the T cells. You could also purify T cells with a density gradient (works great for activated T cell blasts). What might help to seperate the T cells from other cells is long vortexing (I have used this for the seperation from APCs), but it depends on what you want to use the cells for after that. Vortexing could be to harsh. Best of luck!
I think it depends on how you want to analyze the T cells. If you are going to look at their activation markers (say CD25, CD45, CD69) , you can tell whether the cells are one single cell (singlets or doublets) on the flow cytometer. You might not even need to obtain a pure t-cell population. Then you should take care in how you remove the cells from the co-culture. Do not use trypsin. This will cleave away some markers and affect your result. Using a cell dissociation buffer or the suggestions by Ty Dale should work.
If you plan to use the T-cells that have been exposed to adipocytes for future experiments (i.e keep them in culture), then I guess you can try magnetic beads first. And if that doesn't work, cell sorting.
If you have access to a cell sorter I would recommend to label the T cells e.g. with CFSE (the beauty here is that you can simultaneously measure proliferation by flow cytometry) and then either label the other cell population with a different dye or gate on CFSE+ singlets in the FACS (using e.g. FSC-A vs FSC-H and SSC-A vs SSC-H) for sorting. Another option would be to isolate both cell populations from congenic mice differing in the expression of one allel (e.g. CD90.1 and CD90.2) and afterwards use these differences for either MACS-based or FACS-based separation...
Isolation T cell from co-culture is easy as mention above. For me important thing is what is best condition to have any effect on T cell. You need to know what is ratio or fluency to T cell ratio for the effects and how long it will take etc. Any effect for T cell effect on Adipocyte death or make them floating etc. It will have effects on T cell as well Have you done optimisation for this co-culture?
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