We are stimulating T lymphocytes using a coating of aCD3 and aCD28 on p96 wells, but we have difficulties to detach them after 24 hours. Does anybody have an idea of how to achieve that? We need them alive in order to co-culture them with another cell population. Thank you in advance!
The classic method would be an enymatic digest with trysion-EDTA, tryLE ect http://www.lifetechnologies.com/at/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/trypsin.html) but this method can have effects on the expressed epitopes of the cell in case this is important for your T-cells. An NON enzymatic alternative is Versene (EDTA only http://www.lifetechnologies.com/order/catalog/product/15040066) or a simple 10 or 5 mM EDTA solution in 1xDPBS (without MgCl2 and CaCl2) to gently detach them. This let the surface structures intact but harms Mg and Ca dependent surface receptors or epitopes, because EDTA is complexing them. Today I tryed 10 mM EDTA in 1xDPBS with 1% FCS to detach 4 cell lines and the best was for HeLa, after 6 min in the 37°C cell incubator I got a nearly comparable single cell suspension to trypsinized cells. MDA MB468 14 min and A549 8 min but showed much more 4 or more cell aggregates, H1975 was only to loosen by tapping the flask against the table plate, maybe the incubation time should be increased. But for an initial flow cytometry test it was a good result. I hope this helps you.
Hello Carolina,
Hi Volker,
Thank you for your kind answer. We will try the second option you've proposed.
Cheers!
After trying trypsin and EDTA, we ended up just mechanically lifting them with a cell scraper (surface expression was important for our experiments). The surface expression can still be diminished (can some molecules literally pop out of the membrane?) but it works surprisingly well for T cells. You can see if it is working. I think it would be a challenge in a 96 well plate, but perhaps you can redesign the experiment - or else give it a try and see what the yield is like. I can't remember if we washed them first and added Ca/Mg- PBS to encourage them to lift up? Quite possible - I'd try them both, depending on what your subsequent step is.
Thank you for your answer Lisa. Other times I used to detach them adding cold PBS two or three times, and it worked out. But the problem with my current design is than I need to mantain the concentrations of the molecules in the medium of T-cells without diluting in PBS, so I can use the T-cells in their 24h-culture medium for the subsequent steps. Anyway, I'll think about the options you propose!
Hi Carolina.
You should try BD FastImmune EDTA or PBS-EDTA to recover all activated cells.
Hi Carolina,
due to your experimental design a would recommend a gently cell scraping. Sounds rigorously, but I had the best experience with this method for my B-cells... We have small scrapers from TPP and cut the sides manually to fit them for 96-wells...
You should not need to use EDTA extra....place the plates on ice and leave them for a few minutes then simply pipette up and down a few times and all T cells should detach if in clusters. You can do an additional wash of the wells with cold PBS to remove any remaining ones, spin down the PBS/cell suspension to pellet the cells, remove the PBS and resuspend with your cells in culture media so nothing become diluted.
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