Thisprotocolisbasedondifferentialcentrifugationthatallowstoseparateplateletsfrombloodcells.AlsoyoucanuseRobinsonprocedure(consulttheHANDBOOKOFFLOWCYTOMETRYMETHODS)

PROTOCOL

1. Bleedanimalsviatheplexusretroorbitalisorthetailveinunderetheranesthesia
2. Collectbloodinto1,5EppendorftubeswithACD(acid-citrate-dextrose;SigmaC-3821)=1volofACD:3volofblood:6.6volofNaN30,05%-PBS
3. Cetrifugeat216-220g,5-7min,10ѓC
4. Take&saveat4ѓCPRP(platelet-richplasma)
5. Add1mlofeitherNaN30,05%-PBSorBSGCtotheremainigeythrocytesandothercells
6. Cetrifugeat216-220g,5-7min,10ѓC
7. Take&saveat4ѓCPRP(platelet-richplasma)
8. Add1mlofeitherNaN30,05%-PBSorBSGCtotheremainigeythrocytesandothercells
9. Cetrifugeat216-220g,5-7min,10ѓC
10. Take&saveat4ѓCPRP(platelet-richplasma)
11. PoolsavedPRP
12. Cetrifugeat1613g,10min,10ѓC
13. Aspiratesupernatant(SN)
14. ResUSPendsedimentedplateletsineitherEDTA1%-NaCl0.9%orBSGC
15. Cetrifugeat216-220g,5-7min,10ѓC
16. ResuspendinineitherEDTA1%-NaCl0.9%orBSGC.Countplatelets
17. TransferSNintoanothertube,saveat4ѓC

SOLUTIONS

1. ACD=0.1mol/Ltrisodiumcitrate,0.11mol/Ldextrose,and71mmol/Lcitricacidmonohydrate
2. ACDpH4.9=trisodiumcitratedihydrate22g/L+citricacidmonohydrate8g/L+dextrose24.5g/L
3. NaN30.05%-PBS(for500ml)=100mlPBS(x5)+400mlddH2O+0.25gNaN3pH7.0-7.2
4. PBS(x5)(phosphatebuffersalinex5)(for5L)=180gNaCl+37gNa2HPO4*2H2O+10.75gKH2PO4+ddHOto5L
5. EDTA1%-NaCl0.9%(for500ml)=5gEDTA+0.9%NaClto500mlpH7.0
6. BSGC=8.6mMNa2HPO4,1.6mMKH2PO4,0.12MNaCl,0.9mMEDTA,13.6mMNacitrate,11.1mMglucose,pH7.3(Levinetal,1999)

ADDITIONALINFO

1. it"spossibletobleedmicebycardiacpunctureunderetheranesthesia.
2. it"spossibletocollectbloodintosodiumcitrate3.8%(0.1mol/L)=1volcitrate:9volblood:50volbufferedsaline-glucose-citratepH7.3//orheparin7.5U/ml
3. RCF=1.12xrx(RPM/100)x(RPM/100)whereRCF-relativecentrifugalforceing,r-centrifugerADIusinmm,RPM-rotationspermininrpm
4. ToavoidcontaminationofPRPwithleukocytesanderythrocytestakePRP-upper&middlelayers

REFERENCES

1. Semple,J.W.,Speck,E.R.,Cosgrave,D.,Lazarus,A.H.,Blanchette,V.S.andFreedman,J.(1999).ExtremeleukoreductionofmajorhistocompatibilitycomplexclassIIpositiveBcellsenhancesallogeneicplateletimmunity.Blood93(2):713-20.
2. Jin,J.,Quinton,T.M.,Zhang,J.,Rittenhouse,S.E.andKunapuli,S.P.(2002).Adenosinediphosphate(ADP)-inducedthromboxaneA(2)generationinhumanplateletsrequirescoordinatedsignalingthroughintegrinalpha(IIb)beta(3)andADPreceptors.Blood99(1):193-8.
3. Khetawat,G.,Faraday,N.,Nealen,M.L.,Vijayan,K.V.,Bolton,E.,Noga,S.J.andBray,P.F.(2000).Humanmegakaryocytesandplateletscontaintheestrogenreceptorbetaandandrogenreceptor(AR):testosteroneregulatesARexpression.Blood95(7):2289-96.

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