0100,0100,0100On 9 Nov 2002, at 11:47, Finney, Simon wrote: 7F00,0000,0000> > Dear All, > > As a rather minor part of someones project I would like to isolate a > relatively pure preparation of rat granulocytes, stimulate them ex vivo, and > then stain for surface markers/ F actin. We wanted to stimulate a pur(ish) > population. I found a simple gradient system for purification called NIM2 from > Cardinal Associates on the net but their telephone number is unobtainable - does > any one know who supplies it nowadays or alternatively of a simple purification > method. Here is a section from my ancient "Handbook of Flow Cytometry Methods" which is totally out of date but anyway, I found the word file from late last century and here is what I suggest - this methods yields more neutrophils that you could possibly serve at a dinner-party of bacteria. Paul Robinson >From the Handbook of "Flow Cytometry Methods" somewhere about page 177.... ------------------------------------------------------------------------------------------------- outTimes(7) Obtaining Rat Peritoneal Neutrophils(7) Obtaining Rat Peritoneal Neutrophilso Outline  Rat peritoneal neutrophils are an excellent source of aCourier Timeshomogeneous population of immune cells and can be obtained in vast numbers. The cells are elicited in the peritoneum by injecting a solution of 1% glycogen into the peritoneal cavity and washing cells out 4 to 5 hours later. It is important to realize that these cells are not unstimulated or latent but highly stimulated. They can, however, be further stimulated by agonists such as PMA. Once these cells are isolated and washed, keep them on ice at all times. outMaterials leftSyringes: 1 ml (TB) with 25G, 5/8 inch needle; 60 ml with 18G, 1 1/2 inch needle. left1% glycogen: Keep refrigerated (good for about 1 month). Warm glycogen before use if possible. left120 ml saline (or PBS) + 0.1 - 0.2 ml heparin (1000 U/ml). leftWide-mouth beaker with 2 single-thickness gauze draped over top. leftPBS gel (pH 7.4) or HBSS to rinse peritoneum. Keep on ice. leftSurgical instruments. outProcedure outNote: It is important to prepare cells quickly. out1. Anesthetize 0.5 - 0.8 ml ketamineketamine (100 mg/ml). Inject intramuscularly using 25G, 5/8 inch needle and 1 ml (TB) syringe. out2. 50 ml warmed glycogenglycogen into the peritoneum using 18G, 1 1/2 inch needle, bevel up. Feel the tip of the needle through the skin. The glycogen can be felt flowing out into the cavity. If a lump starts to form, the needle is improperly positioned. Reposition needle. out3. Wait 4 to 6 hours, no less and no more. out4. Re-anesthetize rat, as above. The ketamine injection may need to be supplemented before surgery. out5. Inject 40 ml heparinized saline and massage intestines. Refill syringe with heparinized saline. out6. Make 3 cm incision along ventral midline through peritoneal wall. Hold rat over gauzed beaker and drain PMN-rich saline. Rinse peritoneum with remaining saline. If intestinal bacterial contamination has occurred (i.e. intestine has been severed), PMNs will be unusable. out7. Immediately pour cells into 50 ml tubes and centrifuge for 8 minutes at 400 x g. out8. Wash cells 2 times with cold PBS gel or HBSS. out9. Dilute cells to desired concentration and leave on ice until ready to use. out10. Sacrifice rat with appropriate methods out-------------------------------------------------------------------------------------------.Courier 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