FAQ  •  Register  •  Login

Autosampler Sample Integrity

<<

robs

Contributor

Posts: 40

Joined: Mon Dec 02, 2013 3:42 pm

Location: University of Connecticut

Post Thu Apr 03, 2014 3:30 pm

Autosampler Sample Integrity

Hi All,
I'm looking for some feedback from people with autosamplers and/or have samples that remain in DI water for extended periods.
We are getting the autosampler soon for our CyTOF2 and one concern we have is how the cells will hold up being suspended in water for several hours as the plate is run. Usually we leave our samples in PBS and wash in water only until the CyTOF is immediately ready for the sample. Otherwise even fixed cells seem to start coming apart over time, increasing background. We saw this on cells fixed with formaldehyde (Cytofix/perm) and PFA (1.5%, 2%, and 4%). 10-20 minutes seems fine but more than that is a problem.
Are there any other cautions that should be taken with the samples? e.g. use low binding plates or use certain well shapes.
Thanks for your insightful comments,
Rob
<<

mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Apr 03, 2014 4:24 pm

Re: Autosampler Sample Integrity

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.
<<

Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Fri Apr 04, 2014 7:18 am

Re: Autosampler Sample Integrity

As Mike noted, Ir will diffuse out over time. We minimize this issue by keeping the cells in a very small volume of water until just before the run. Typically 1x10^6 cells in 50ul or so (not precisely measured at this point). When comparing signal decline of beads vs. Ir there is no real difference using this procedure.
I did not see cell loss over time when 1.6% PFA-fixed cells were in water for as long as 5h (haven't tested more).
<<

robs

Contributor

Posts: 40

Joined: Mon Dec 02, 2013 3:42 pm

Location: University of Connecticut

Post Tue Apr 08, 2014 2:02 pm

Re: Autosampler Sample Integrity

Thank you both, this puts my concerns aside. When I brought this topic up at a local user meeting yesterday they reiterated your advice/cautions.

Return to CyTOF general discussion

Who is online

Users browsing this forum: No registered users and 18 guests