OUTLINE In order to avoid platelet activation all manipulations must be performed as quickly and as acurate as possible.Work on ice if possible!

This protocol is based on differential centrifugation that allows to separate platelets from blood cells. Also you can use Robinson procedure (consult the HANDBOOK OF FLOW CYTOMETRY METHODS)

PROTOCOL

Bleed animals via the plexus retroorbitalis or the tail vein under ether anesthesia Collect blood into 1,5 eppendorf tubes with ACD (acid-citrate-dextrose; Sigma C-3821) = 1 vol of ACD : 3 vol of blood : 6. 6 vol of NaN 3 0,05%-PBS Cetrifuge at 216-220g , 5-7min , 10ѓC  Take & save at 4ѓC PRP (platelet-rich plasma )  Add 1ml of  either NaN3 0,05%-PBS or BSGC to the remainig eythrocytes and other cells Cetrifuge at 216-220g , 5-7min , 10ѓC Take & save at 4ѓC PRP (platelet-rich plasma ) Add 1ml of either NaN3 0,05%-PBS or BSGC to the remainig eythrocytes and other cells Cetrifuge at 216-220g , 5-7min , 10ѓC Take & save at 4ѓC PRP (platelet-rich plasma ) Pool saved PRP Cetrifuge at 1613g , 10min , 10ѓC Aspirate supernatant (SN) Resuspend sedimented platelets in either EDTA 1%-NaCl 0.9% or BSGC Cetrifuge at 216-220g , 5-7min , 10ѓC Resuspend in in either EDTA 1%-NaCl 0.9% or BSGC. Count platelets Transfer SN into another tube, save at 4ѓC

SOLUTIONS

ACD = 0. 1 mol/L trisodium citrate, 0. 11 mol/L dextrose, and 71 mmol/L citric acid monohydrate  ACD pH 4. 9 = trisodium citrate dihydrate 22g/L + citric acid monohydrate 8g/L + dextrose 24.5 g/L NaN3 0.05%-PBS (for 500ml) = 100ml PBS(x 5) + 400ml ddH2 O + 0.25g NaN 3 pH7.0-7.2  PBS (x5)(phosphate buffer saline x5) (for 5L) = 180g NaCl + 37g Na 2 HPO4 *2H 2 O + 10.75g KH2 PO4 + ddHO to 5L EDTA 1%-NaCl 0.9% (for 500ml)= 5g EDTA + 0.9% NaCl to 500ml pH 7.0 BSGC = 8.6 mM Na2HPO4, 1.6 mM KH2PO4, 0.12 M NaCl, 0.9 mM EDTA, 13.6 mM Na citrate, 11.1 mM glucose, pH 7.3 (Levin et al, 1999)

ADDITIONAL INFO

it's possible to bleed mice by cardiac puncture under ether anesthesia. it's possible to collect blood into sodium citrate 3.8% ( 0.1 mol/L ) = 1 vol citrate : 9 vol blood : 50 vol buffered saline-glucose-citrate pH7.3 // or heparin 7.5 U/ml     RCF = 1.12 x r x (RPM/100) x (RPM/100) where RCF-relative centrifugal force in g   , r -centrifuge radius in mm , RPM-rotations per min in rpm To avoid contamination of PRP with leukocytes and erythrocytes take PRP-upper&middle layers

SCHEME REFERENCES

Semple, J.W., Speck, E.R., Cosgrave, D., Lazarus, A.H., Blanchette, V.S. and Freedman, J. (1999). Extreme leukoreduction of major histocompatibility complex class II positive B cells enhances allogeneic platelet immunity . Blood 93(2): 713-20. Jin, J., Quinton, T.M., Zhang, J., Rittenhouse, S.E. and Kunapuli, S.P. (2002). Adenosine diphosphate (ADP)-induced thromboxane A(2) generation in human platelets requires coordinated signaling through integrin alpha(IIb)beta(3) and ADP receptors . Blood 99(1): 193-8. Khetawat, G., Faraday, N., Nealen, M.L., Vijayan, K.V., Bolton, E., Noga, S.J. and Bray, P.F. (2000). Human megakaryocytes and platelets contain the estrogen receptor beta and androgen receptor (AR): testosterone regulates AR expression . Blood 95(7): 2289-96.