Hi gry

Isolation from Blood coagulated with Heparin or EDTA??

please find the link.

http://link.springer.com/article/10.1007%2FBF00368459

We have used EDTA - thanks for the link :)

Blood was collected from the ulnar vein of chickens into disodium ethylenediaminetetraacetic acid (EDTA) Vacutainer tubes (Fisher Scientific, Norcross, GA). Heterophils were isolated using modified techniques of published procedures (Madyastha et al. 1982; Latimer et al. 1989; Andreasen et al. 1991). Disodium EDTAanticoagulated blood was mixed with 1% methylcellulose, in calcium- and magnesium-free (CMF) Hank's balanced salt solution (HBSS; Sigma Chemical Co., St Louis, MO), 1% fetal bovine serum (FBS), at a 1.5:1 ratio and centrifuged for 15 min at 25 g. The extended huffy coat and plasma were recovered and washed once in CMF HBSS 1% FBS. The samples were resuspended in the same solution, and 3 ml was carefully layered over a preconstructed discontinuous Histopaque 1.077/1.119 (3 ml over 3 ml) density gradient (Sigma). The gradients were centrifuged for 20 minutes at 225 g. The buffer, bully coat at the buffer/1.077 interface, and 1.077 layers were removed and discarded. The remaining 1.119 layers and call pellet were resuspended in CMF HBSS I%FBS. After one wash, the cell pellet was resuspended in seven volumes of 0.87% NH4C1 in 0.1% KHCO3 and rocked for 10 min to lyse erythrocytes. The remaining heterophils were washed twice as before. Neutrophils were isolated similarly but without the need for the methylcellulose step (necessary to separate out nucleated avian erythrocytes). Both heterophil and neutrophil purity and viability were routinely verified. Purity was verified visually from cytocentrifuge preparations and was consistently >95%, with few of the contaminating cells being leucocytes. Cell viability was consistently >95% as well, as determined by exclusion of trypan blue dye. Incubation temperature for heterophils was 41°C and for neutrophils was 37°C, consistent with host body temperature.