Ray: I prefer that you reverse this proceedure. If you first run the bllod on ficol and remove the top layers, you are left with basically PMN and RBC. Then you can mix this layer with dextran T500 and leave it for 37 deg and remove the 'buffy" coat. You will want to do a quick rbc lysis and resuspend in saline with some glucose and other goodies to make the pmn happy, but not ecstatic. There are numerous other isolation proceedures depending on how many cells you need. The process above is the way to get lots of cells. There are less activating ways for smaller numbers of cells including using the NIM centrifugation method mentioned by others recently. You need to do this as quickly as possible as PMN are simply not happy living very long! The PMA will activate the respiratory burst of PMN every time. regards paul robinson Purdue university On 15 Nov 2002, at 15:25, ray hester wrote: > > An investigator here is comparing the effects of activated [using phorbol] > vs non-activated human PMN, but whereas they expected (based on other > studies) the activated PMN to have an enhancing effect compared to the > unactivated PMN, they find similar results from the addition of either > population. > > Their PMN isolation procedure involves dilution of blood 1:2 with saline, > addition of dextrin to 5%, allowing cells to settle for 45 min at 37 C, > taking the supernate and underlaying it with Histopaque, and centrifuging > for 30 min (I don't know the rpm). The PMN are recovered as the pellet. [I > believe I've described this accurately] > > The markers they are looking at, on human red blood cells, are percentages > of RBC expressing CD36, CD49, and phosphatidyl serine. The activated PMN > were expected to increase these values compared to unactivated, i.e., non > PMA treated, PMN, when incubated with the RBC. > > Would the isolation procedure described above be expected to activate PMN > without the addition of PMA? > > If so, does anyone know of a gentler isolation procedure which might > not/does not activate PMN? > > Thanks for any thoughts. > > Ray > > Ray Hester > Univ. of South Alabama > Mobile, AL 36688 > rhester@jaguar1.usouthal.edu > J.Paul Robinson, PhD PH:(765)4940757 Professor of Immunopharmacology Professor of Biomedical Engineering Purdue University FAX:(765)4940517 EMAIL:jpr@flowcyt.cyto.purdue.edu WEB: http://www.cyto.purdue.edu
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