Hi Keith; We have published a few papers on whole blood analysis of PMN function. The first was: Simon, S.I., Chambers, D.C., Butcher, E., Sklar, L.A. (1992) Neutrophil aggregation is b2 Integrin and L-selectin dependent in blood and isolated cells as measured by flow cytometry. J.Immun. 149:2765-2771. & Kostantinos Konstantopoulos, Neelamegham S., Burns, A.R. Kansas, G.S., Snapp, K.R., Berg. E.L., Hellums, J.D., McIntire, L.V., Simon, S.I., (1998) Venous levels of shear induce neutrophil-platelet and neutrophil aggregation in blood via P-selectin and ß2integrin. Circulation 98: 873-882. Hope this helps Scott Simon At 09:24 AM 11/27/02 +0100, Keith Thompson wrote: >Thanks for the information. Sorry to trouble you more, but do you have >details or a reference to the 'whole blood system'? > >Dr K.Thompson > >IMMI >Oslo >Norway > >----- Original Message ----- >From: "Arnold Richard Pizzey" <a.pizzey@ucl.ac.uk> >To: cyto-inbox >Sent: Tuesday, November 26, 2002 12:20 PM >Subject: PMN isolation w/o activation > > > >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 >_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/ Scott I Simon, Ph.D. Professor of Biomedical Engineering University of California, Davis One Shields Avenue 1025 Academic Surge Davis, CA 95616-5294 530-752-0299 Office 530-752-1430 Lab 530-754-5739 Fax 530-759-4380 Pager sisimon@ucdavis.edu http://www.bme.ucdavis.edu/faculty/ScottISimon.html
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:31 EST