I'm isolating mouse PBMCs for an ELISpot assay. I work as quickly as possible but the cells do sit for 60-90 min while I'm counting cells and getting everything ready for the assay. The PBMC are in serum free media in 15mL conical tubes. Should I put these samples on ice or should I place them in the incubator at 37C and 5-9% CO2 with the screw caps loosened?
Yes. They might seriously freak out in the warm. If you're measuring cytokines, they'll almost certainly start secreting stuff in culture that you wouldn't be seeing if they came fresh out of the mouse.
Dear Derek,
Most often we isolate mouse spleen cells and not PBMCs for the ELISPOT. Concerning spleen cells, we always use fresh cells (use within 8h after the isolation of the spleen). In this way, the analyses work perfectly. In the past we have also isolated PBMCs from mouse. We found that it was critical to keep the cells at room temperature and in RPMI-1640 + 10% FCS before use. In this way we did not find much problems with the viability of cells. However, keeping the cells on ice was detrimental for the viability of the cells (this counts for both spleen cells and PBMCs). We realize that serum-free medium is recommended for the ELISPOT assay without pre-stimulation, but incubation of the cells in RPMI-1640 + 10% FCS works fine although the size of the spots becomes a bit smaller.
Thank you for the feedback all. I think I may isolate some cells this week and compare ice/RT/37C. My logic was that we intuitively place cells on ice, but for performing the assay we place at 37C. I will report my results.
We also support saving the cells either at room temperature or at 37C/5-9% CO2 before use. To minimize the risk of having secreted products in the medium it is wise to subject the cells to a washing step before adding them to the plate.
Try using an automated cell counting method to speed up your prep process and reduce the time the cells need to wait..
Hi Derek,
I was wondering if you had any updates on how to store your samples for ELISPOT. I'm in the process of setting up ELISPOT in our lab for antigen specific B cells, and wanted to know if you had any advice on how you stored your cells.
Thanks!
Roopali
Just saw this. There is a paper by ImmunoSpot available on their website that gives some details. Seems that placing loose cap tubes in the incubator works best.
Hi Derek, we usually run ELISPOT assays for PBMCs in serum free medium and is always best to keep your cells warm (37C/5% CO2 before use). We take samples for counting and return the cells to the incubator up to the moment of assay setup. Counting is perform in a flow cytometer to speed up the process when sample number is high.
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