I am writing to ask for help.
I am using Miltenyi anti-NKp46-biotin-bead- MS column kit. The first step is
to get splenocyte from spleen by gentlMACS without enzyme treatment. A very
simple protocol, just put spleen into the PEB buffer and let gentleMACS run
m_spleen_01 program once, and filter, spin, aspirate, resuspend the pellet.
The problem comes from the re-suspending process. The small tight pellet will
produce(or contain) big clump that is almost impossible to break when 10 ml
PEB buffer is added. This clump, when do trypan stain, could see black "nuc
lear" structure, but seems not the cell aggregation. It looks more like fat o
r protein aggregates. Cell count indicate the cell # in the cell suspension
is similar comparing before and after spin.
PEB buffer contains 1xPBS, 0.5% BSA, 2mM EDTA, which is from Miltenyi. I mea
sured the PH, 7.1, very close to the claimed 7.2. I prepare similar PEB buffer, with PH 7.6, using 0.5% FBS as a substitution of BSA, and got similar clumps.
I have tried room temp and 4 degree, both produce similar clumps. If filter
these clumps and spin the pass through again, I will get suspension with clumps again (but today, after leaving the suspension from first spin on ice for half day, the 2nd spin did not produce unbreakable clumps anymore)
It is very weird experience. I did get a little clump in the first couple of
rounds, but recently, it get very big and obvious. The difference I can tell is only that recent spleen is from sick mouse, while the initial experiments use CD1 wild type mouse.
This is a long post. Sorry. But I have called Miltenyi several times. They d
on't have clue either. (Buffer is the concern, but obviously it is not the cause; It is just put 75 ml BSA to 1475 ml rinse solution, both of which are from Miltenyi). Anybody have suggestions? I need to get this protocol work d
uring Christmas break. .......Thanks a lot.
problems with my spleen single cell suspension by gentleMACS
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