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 www.tcd.ie/biochemistry/flow ----------------------------------------- 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… ----------------------------------------- I sort DCs on an Aria using the 100um nozzle and 20 psi or whatever "low" really is. They look fine post-sort. ----------------------------------------- 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 ---------------------------------------- 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. ----------------------------------------- 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. ----------------------------------------- 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 ----------------------------------------- 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. ----------------------------------------- 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. ----------------------------------------- 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.Received on Wed Apr 19 13:38:00 2006
This archive was generated by hypermail 2.1.8 : Fri Apr 21 2006 - 04:12:02 EDT
