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Many thanks to all for their very helpful responses- I thought I'd just
include the summaries below as it may help others... For me, dropping the
pressure (though keeping the same 70um nozzle- waiting on delivery of 100um
one) seemed to do the trick- at 25psi we saw no activation compared to
controls. Am awaiting LPS test results for the sheath lines etc...
Cheers
Barry.
Barry Moran
Flow Cytometry Facility
School of Biochemistry and Immunology
0.19, Biotechnology Building
Trinity College Dublin
(01) 608 3575
barry.moran@tcd.ie
http://www.tcd.ie/biochemistry/flow
Question about:
Dendritic cell activation during sort
Responses:
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Hi. I think just the process of sorting is activating your cells. You may
have to resort to MACS negative selection to enrich for your populations…
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I sort DCs on an Aria using the 100um nozzle and 20 psi or whatever "low"
really is.
They look fine post-sort.
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I have had researchers tell me that their DCs have been activated following
a sort at the parameters you indicated.
However, this can be avoided by dropping the sheath pressure.
There is significantly less activation at 35psi (70 um nozzle, but 85um
nozzle may be better!), and almost none at 20psi (100um nozzle). I do have
one researcher who would like me to investigate sub 20psi values if I can
persuade the FACSAria to play ball!
Regards
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For a long time I’ve been doing a lot of DC sorting on my MoFlo, always at
30 – 32 psi on a 90µ tip, and they’re healthy and well post-sort, with no
signs of activation. We found in the past that many activate and/or die
after high-pressure (60 psi/70µ) sorting.
They are large enough cells and seemingly sensitive enough to the higher
sort pressures that I always “default” to the larger tip and lower pressure
for DC sorting. We sort a lot of naïve DCs this way for transfer
experiments and end up with great numbers of “healthy” DCs.
Try this set up. I hope it works for you. Any other questions, give me a
shout.
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We have found exactly the same thing when sorting cultured murine DCs on our
Vantage SE. However, my 'customer' had the forethought to check whether the
activation was due to the sorting or the cell preparation step. She tested
this by preping her cells and, just before giving them to me for sorting,
she took a small aliquot and put them straight back into culture. After
sorting the resultant cells were also put into culture. She then checked
both lots of cells and found BOTH to show the same degree of activation.
Hence, we concluded that the sorting wasn't the problem, preping the cells
was, and no matter what we did with our sorter the cells were always going
to show some degree of activation. Have you checked that this isn't the case
with your cells? If you have, then I been told that thoroughly cleaining the
sheath lines sample lines etc and using endotoxin free PBS as sheath is
advisable, but I don't know whether it works.
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Hey Barry
You should probably lower your pressure and increase your nozzle size.
I would imagine quite a few cells perish when blasted out of the nozzle.
Under 20psi and a 100u nozzle would be kinder to your cells.
Best
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I have heard many people here say that if you even pipet dendritic cells too
vigorously you see some activation, so I would not be at all surprised that
sorting could do the same thing. All I can think of would be to run slower,
with lower pressure.
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The DCs could very well be getting activated by LPS present in your system.
Macrophages can also be sensitive to the presence of LPS.
There are microtiter plate based tests that you can use to measure LPS
levels in your system (we use the LAL QC1000 test from Cambrex).
We have been monitoring LPS levels on our MoFlo for over 2 years now and
have found the in-line sheath filter to be the greatest source of LPS
contamination. For prevention, we take care to dump out whatever is
remaining in the sheath tank and rinse it with 70% EtOH, as well as flush
the lines with 70% EtOH every evening. We also periodically "soak" the
inside of the sheath tank with 5 N NaOH to destroy any LPS present there and
change the sample line in addition to the in-line sheath filter. If you need
any more details you can contact me off- line.
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I have had the activation problem earlier in my sorter life, and the trick
is to be as gentle to the cells as possible. So I suggest that they make
sure that their controls are treated the same way as the sorted cells
regarding washes, centrifugation and so on, to rule out that it is related
to the preparation.
If that rules out the prep as the cause, then try to diminish the pressure
on the MoFlo and sort with the lowest possible pressure, and sort into 'wet'
coated tubes.
