[Cytometry] Sorting Ethanol fixed cells - high conflict, low efficiency

Hassel, Christiane A. chassel at indiana.edu
Fri Feb 28 14:26:26 EST 2014


Hello all,

I checked the listserv for previous post - there was one post with one answer from 2002;  thought I'd ask this question anyway in case there has been additional insight over the last 12 years.

I have a researcher who I am helping sort ethanol fixed tissue culture cells, DAPI labeled, for G1 and G2/M populations for subsequent Western blotting.  For many of the samples, I am seeing a high conflict/low efficiency (30-50% efficiency).  We also see an erratic threshold count (also evident when looking at Time parameter) for some of these samples.  In some of the samples there is quite a bit of subG1, but other samples we see very little subG1 (different cell lines).  The goal is to try and decrease the conflicts and increase the efficiency.  Any advice is greatly appreciated.  Here's some additional info:

Aria II sorter, 130um nozzle, 13psi (we tried with 100um at 20psi, resulted in side stream fanning)
Window Extension at 4
Event rate no more than ~2000-3000 events/sec
Samples in 1x PBS, kept on ice, covered from light (more recently samples in 1x PBS 0.1% Triton X-100 + 1% BSA + EDTA + RNAse)
Purity sorting
Area scaling adjusted
Threshold on DAPI (10,000); first DAPI peak at 60,000 (for one cell line G1 and G2/M very well resolved, another cell line where there is quite a bit of subG1, subG1 and G1 not well resolved)
Violet laser - 50mW
Samples filtered before sorting and don't appear "chunky"
After 70% ethanol fixation, sample is kept at -80 until ready to stain (research mentioned samples appear viscous - one sample appeared frozen)

Thanks in advance for any help,

Christiane


Christiane Hassel, MS
Manager, Flow Cytometry Core Facility
1001 E 3rd Street Jordan Hall 029
Bloomington, IN 47405
Phone: 812-855-7101
E-mail: chassel at indiana.edu<mailto:chassel at indiana.edu>
Website: facs.bio.indiana.edu
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