Langerhans Cells and Sorting

Hi there Flow People,

Have any of you been sorting a population of cells ­ for instance,
Langerhans Cells from the skin of mice stained with MHC Class II-FITC to
identify them from keratinocytes in a single cell skin suspension - and
observed a type of Œflashing¹ of signal intensity every few seconds?

In numerical terms, this translates to a jump up (transient) in percentage
of cells inside sort gate from 1.5% to 15 -30%. Visually, this looks like
the cells are dancing up and down the FL-1 axis in time to the stereo. I
haven¹t sorted langerhans cells before but I have sorted skin preparations
previously and I have not seen this feature. If anyone has any suggestions
for what to do about this, it would be much appreciated.

And a related question: my strategy for purifying these cells has been to
run them through on a Œyield¹ sort mode to increase my target frequency (the
cells are jiving away throughout this sort). Inclusion of a Œsingle cell¹
gate using FSC-height vs FSC-area does not eliminate the jiving. This sort
increases the purity to about 12% which I then re-run through in a high
purity sort mode. In this second sort, the cells are minus the rhythmic
finesse of the first yield sort, with about 1 ­2 logs separation between the
majority of positives and negatives (except for a small proportion of highly
autofluorescent cells) and the majority of them are Œsingle¹ events however
the resulting purity is only as good as 60% of events in the Œlive cell
gate¹ in FSC/SSC. I re-checked my drop delay and it was spot on so I am
really puzzled with what might be wrong here.

Little extra details about my situation - I¹ve got a FACSVantage FACSDiVa
that I used in conjunction with a 100µ nozzle, 20 psi, approx 36k ddf, the
cells are prepared from mouse skin using enzymatic digest along with trypsin
incubation steps to reduce clumping and passed through a 35µ gauze filter
prior to sorting. Sort buffer = freshly made PBS, sample buffer = PBS + 2%
BSA.

Any advice or suggestions, gratefully accepted.

Thanks a lot!
Joanna


--
Joanna Roberts
--
Malaghan Institute of Medical Research
PO Box 7060 Wellington
New Zealand
64 4 499 6914 x 850 (ph)




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