Hello Ray, More years ago than I care to remember, I did a lot of work with PMNs looking at the respiratory burst as well as activation markers. Activation of PMNs was not usually a problem. (Although you will always get the odd individual donor whose PMNs are upregulated) After playing around for a couple of years with different schemes for Granulocyte (remember, you're going to get some Eosinophils in there as well unless you deplete them) isolation, we came up with following: Note all operations are to be carried out at room temperature only, put them at 37' C or on ice and they will upregulate. The assumed volume of blood is 50ml i) Take blood into EDTA. ii) Add 5ml dextran (in 0.9% NaCl) and mix very gently for about 2 minutes. iii) Draw up the above mixture into a syringe with an offset luer, taking care not to introduce bubbles. iv) Place the syringe *on its side* with the luer uppermost and leave for 5mins. vi) After 5mins, sharply tap the syringe and leave for another 5mins. vii) Incline the syringe and expel the supernatent directly onto a cushion of equal volume Ficoll (you can do this with either a kwill filling tube on the end of the syringe, or a large gauge needle) viii) Centrifuge 400g/20mins at room temperature -a refrigerated centrifuge is ideal to maintain the temperature at 20'C. ix) Discard the supernatent by inverting the tube. x) Resuspend in Granulocyte maintenance buffer (PBS +Mg +Ca +5mM Glucose) Cells will be quite happy in this buffer at room temperature for up to about 4-5 hours. I have tried keeping the cells on ice, but generally they seem to do worse. With some practice, you can use the above cells directly -the RBC contamination is minimal however, if you wish, the following hypotonic lysis method give minimal upregulation. Note, all solutions are 0'C i) To the pellet, add 2ml Phosphate buffered saline (no Ca/Mg) and carefully resuspend to an homogenous mixture -avoid pipetting up and down- you can examine the cells at this point to ensure that there are no aggregates. ii) Add 5ml H20 and gently rock the tube for 45 seconds. iii) Add 2ml 3.6% Saline. iv) Add 40ml PBS (no Mg/Ca. v) Centrifuge 200g 10mins at room temp. vi) Resuspend in maintenance buffer (see above) Has your investigator considered moving to a whole blood system? -this might be seen as a step closer to the physiological state. We did quite a lot of work in this system using DCF as the output signal. (although there are better compounds now) -You can stop the burst by addition of 5mM final Conc. NEM then lyse the red cells with immunoprep (Beckman-Coulter) By the by, if you are going to use receptor-mediated activation (say fmlp) you will see **none** as there appears to be an inhibitory factor in plasma which can only be overcome by priming the cells with something like GM-CSF. Best regards, Arnold 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 _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/ Arnold Richard Pizzey Department of Haematology Royal Free and University College London Medical School 98 Chenies Mews London WC1E 6HX U.K voice: +44 020-7679-6234 Fax: +44 020-7679-6222 email: a.pizzey@ucl.ac.uk _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
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