Thu Apr 03, 2014 4:24 pm by mleipold
Hi Rob,
We generally haven't had an issue with cells lysing over a couple hours in water, if they were properly/completely fixed to begin with (2% PFA/PBS, made fresh from a 16% methanol-free stock ampule that has been open less than one month; typically 4C overnight is how we fix for our workflow, but 15-20min at room temp also seems to work ok in most cases like phospho).
We didn't have any noticeable difference between round vs V-bottom. Since V-bottoms are often square wells (at least for deep-well plates), the X/Y alignment can be more forgiving.
The issue we *have* had is increased background, primarily in the Ir channels. Since the intercalation is non-covalent, the intercalator *will* diffuse back out, slowly. Because of the levels used during staining, you typically won't see much of a decrease in your Ir signal intensity, but the channels will have increased streakiness. This is also an issue for some antibodies like CD45/CD45RA and even some CD8 clones, which just seem to have a higher background streakiness even after careful titering.
We also have had some issues with overall cell recovery from autosampler samples. In a head-to-head where the same sample series was run manually and then with the autosampler, the autosampler consistently gave worse recovery. (In this test, increasing cell numbers were plated; I think we varied 200K through 1.5mil).
We *think* this was due to sample settling over time. Talking with DVS, they reprogrammed our machine to do a larger mixing volume (pipetting up and down before injecting), and readjusted the z-axis alignment so it is drawing from closer to the bottom of the well. We just haven't yet gone back and re-run the experiment to see whether this has fixed our problem.
I would suggest that you test that out as well; the cells *will* settle more the longer they're in the wells.
Also, be aware that the calibration beads will increasingly stick to the cells the longer they're co-incubated. The HIMC hasn't rigorously tested this, but the Nolan lab has some info on it. I don't think it's a huge problem (ie, you won't wind up throwing out 50+% of your cells as bead-cell doublets), but it's still something to be aware of.