Hi! I am working on a biophotonics project in which I need isolated but inactive CD4+ splenocytes. Every protocol and piece of literature I have found immediately activates and cultures splenocytes with IL-2 after collection/ isolation in order to mature them, but I do not want my cells to mature. I need them to stay naive. I genuinely have no idea if there is a common method for keeping immature T-cells alive for extended periods of time.
I've been having trouble getting consistent results in my experiments, and I'm thinking that one of the reasons may be that my cells are not healthy (even if they appear alive with trypan blue or PI staining). In order to see the cells respond to my experiments, they do need to be healthy and functioning.
On Monday, all of the cells in a vial were dead. I think that may be because they were left thawed in DMSO for a few seconds too long, but if accidentally leaving them thawed for an extra minute kills all of them, it seems like even if they were removed earlier they might still be really unhappy because in the process of freezing, thawing, and centrifuging they are going to be thawed in DMSO for a little while no matter what.
What I have been doing is freezing the cells in freezing media (DMSO solution) in -20C immediately after they have been harvested from the spleen of a fresh mouse and purified to just the CD4 cells. The longest cells have been left frozen is ~1 month. To thaw, I've been melting the cells in a 37C dry bath, quickly doubling the volume of the vial with warm culture medium, centrifuging 1000xg for a minute, and reconstituting the cell pellet in just culture media. The cells in culture media have been left to incubate in a 37C, 5% CO2 incubator for at least 1 hour but up to ~48hrs. When I use the cells, I dye them before plating but keep them warm. The dye needs an hour to set and some cells may not be used for several more hours while I'm working on other plates. I usually check to see if the cells are alive before dying and there are usually very few dead ones. The plated cells are not responding the way I want them to, but there may be several reasons for this (that I am working on solving).
Does anyone have any advice on how to make sure my cells are healthy? I only get new animals every few weeks, so I need some way to store them.
Thank you!
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