Separation of Platelets from Whole Blood
来自 : 蚂蚁淘
Purpose
Thisprotocoldescribeshowtoisolatehumanplateletsfromwholeblood.Isolatedplateletsareusedforstaticadhesionassays,forflowchamberassays,flowcytometrymeasurements,etc.
Materials
- Citratebuffer(25gsodimcitrate,8gcitricacid,500mlH2O2).
- ProstaglandinE1(PGE1).
- ACDbuffer(6.25gsodiumcitrate.2H2O,3.1gcitricacidanhidrous,3.4gD-glucosein250mlH2O).
- Hepes-TyrodebufferpH7.4(134mMsodiumchloride,12mMsodiumbicarbonate,2.9mMpotasiumchloride,0.34mMsodiumphosphatemonobasic,1mMmagnesiumchloride(avoidforadhesionassay),5mMHepes,5mMglucose,1%BSA).
Procedure
- Draw45mlbloodintoasyringecontaining5mlcitratebuffer.Optional:Add2micromolarPGE1toavoidplateletactivation.
- Transfertoa50mltubeandcentrifugeat150gfor15minutesatroomtemperature.
- Carefullytransfertheupperphase(PlateletRichPlasma)toa15mltubeandadd1/10volumeofACDanticoagulant.
- Pelletplateletsbycentrifugationat900gfor5minutesatroomtemperature.Note:Aftercentrifugation,supernatantstillcontainsasignificantnumberofplateletsandcanbecollectedforexperiments.
- ResUSPendthepelletin5-10mlHepes-Tyrodebuffer.
Keepplateletsatroomtemperaturethroughouttheexperiment.Freshlyisolatedplateletsshouldbeusedwithin2hours.
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