【樣本要求】

1.樣本類型和采(cai)集

血清(qing)：將(jiang)收集于(yu)(yu)血清(qing)分離管的全血標(biao)本在(zai)室溫(wen)放置(zhi)0.5-2小時或(huo)(huo)4℃過夜，然后1000×g離心20 分鐘，取(qu)上清(qing)即可，或(huo)(huo)將(jiang)上清(qing)置(zhi)于(yu)(yu)僅供科研使用，不(bu)得用于(yu)(yu)臨床診斷。-20℃或(huo)(huo)-80℃保存，但(dan)應避免反復(fu)凍融。

血(xue)漿(jiang)(jiang)：用(yong)(yong)(yong)EDTA或(huo)(huo)(huo)肝(gan)素(su)作為抗(kang)凝劑采集(ji)標(biao)(biao)本，并將(jiang)(jiang)標(biao)(biao)本在采集(ji)后(hou)的(de)30分(fen)(fen)鐘(zhong)(zhong)(zhong)內于(yu)2-8℃ 1000×g離心(xin)(xin)15分(fen)(fen)鐘(zhong)(zhong)(zhong)，取(qu)(qu)上(shang)(shang)清(qing)即(ji)可(ke)(ke)(ke)(ke)(ke)檢測(ce)(ce)，或(huo)(huo)(huo)將(jiang)(jiang)上(shang)(shang)清(qing)置(zhi)(zhi)(zhi)于(yu)-20℃或(huo)(huo)(huo)-80℃保(bao)存(cun)(cun)(cun)，但(dan)應(ying)避免反(fan)復(fu)凍(dong)(dong)(dong)(dong)(dong)融(rong)。組織(zhi)勻(yun)(yun)漿(jiang)(jiang)：用(yong)(yong)(yong)預冷的(de)PBS (0.01M, pH=7.4)沖洗(xi)(xi)(xi)組織(zhi)，去除(chu)殘留血(xue)液（勻(yun)(yun)漿(jiang)(jiang)中(zhong)裂(lie)解(jie)(jie)(jie)的(de)紅(hong)細(xi)(xi)(xi)胞(bao)(bao)會影響測(ce)(ce)量(liang)(liang)結果），稱重后(hou)將(jiang)(jiang)剪碎的(de)組織(zhi)與對(dui)(dui)應(ying)體(ti)積的(de)PBS（一般按(an)1:9的(de)重量(liang)(liang)體(ti)積比(bi)，比(bi)如1g的(de)組織(zhi)樣(yang)(yang)品(pin)(pin)對(dui)(dui)應(ying)9mL的(de)PBS，具(ju)體(ti)體(ti)積可(ke)(ke)(ke)(ke)(ke)根(gen)據實驗需要適(shi)當調整(zheng)，并做好記錄。推(tui)薦在PBS中(zhong)加(jia)(jia)入(ru)(ru)蛋(dan)白酶抑制(zhi)劑）加(jia)(jia)入(ru)(ru)玻(bo)璃勻(yun)(yun)漿(jiang)(jiang)器(qi)中(zhong)，于(yu)冰上(shang)(shang)充(chong)分(fen)(fen)研磨。為了進(jin)一步裂(lie)解(jie)(jie)(jie)組織(zhi)細(xi)(xi)(xi)胞(bao)(bao)，可(ke)(ke)(ke)(ke)(ke)以對(dui)(dui)勻(yun)(yun)漿(jiang)(jiang)液進(jin)行超聲破碎，或(huo)(huo)(huo)反(fan)復(fu)凍(dong)(dong)(dong)(dong)(dong)融(rong)。最后(hou)將(jiang)(jiang)勻(yun)(yun)漿(jiang)(jiang)液于(yu)5000×g離心(xin)(xin)5~10分(fen)(fen)鐘(zhong)(zhong)(zhong)，取(qu)(qu)上(shang)(shang)清(qing)即(ji)可(ke)(ke)(ke)(ke)(ke)檢測(ce)(ce)，或(huo)(huo)(huo)將(jiang)(jiang)上(shang)(shang)清(qing)置(zhi)(zhi)(zhi)于(yu)-20℃或(huo)(huo)(huo)-80℃保(bao)存(cun)(cun)(cun)，但(dan)應(ying)避免反(fan)復(fu)凍(dong)(dong)(dong)(dong)(dong)融(rong)。細(xi)(xi)(xi)胞(bao)(bao)裂(lie)解(jie)(jie)(jie)液: 吸(xi)棄培(pei)養液，用(yong)(yong)(yong) PBS（0.01 M，pH 7.4）將(jiang)(jiang)細(xi)(xi)(xi)胞(bao)(bao)洗(xi)(xi)(xi)一遍(bian)。用(yong)(yong)(yong)細(xi)(xi)(xi)胞(bao)(bao)刮刮下細(xi)(xi)(xi)胞(bao)(bao)（不能用(yong)(yong)(yong)胰酶和 EDTA 處(chu)理(li)），加(jia)(jia)入(ru)(ru) 2-5mL PBS（0.01 M，pH 7.4）。懸(xuan)(xuan)浮細(xi)(xi)(xi)胞(bao)(bao)可(ke)(ke)(ke)(ke)(ke)省(sheng)略(lve)。收集(ji)細(xi)(xi)(xi)胞(bao)(bao)懸(xuan)(xuan)液，4℃1000×g 離心(xin)(xin) 10min，棄去培(pei)養基，用(yong)(yong)(yong)預冷的(de) PBS 潤洗(xi)(xi)(xi) 3 次(ci)。加(jia)(jia)入(ru)(ru)適(shi)量(liang)(liang)的(de)預冷 PBS 或(huo)(huo)(huo)非變性細(xi)(xi)(xi)胞(bao)(bao)裂(lie)解(jie)(jie)(jie)液（臨(lin)用(yong)(yong)(yong)前加(jia)(jia)入(ru)(ru)蛋(dan)白酶抑制(zhi)劑）重懸(xuan)(xuan)細(xi)(xi)(xi)胞(bao)(bao),通常 6 孔(kong)板一個(ge)孔(kong)的(de)細(xi)(xi)(xi)胞(bao)(bao)量(liang)(liang)需要 150-250μL PBS 重懸(xuan)(xuan)。 將(jiang)(jiang)樣(yang)(yang)品(pin)(pin)放入(ru)(ru)-20℃或(huo)(huo)(huo)-80℃，使(shi)樣(yang)(yang)品(pin)(pin)冷凍(dong)(dong)(dong)(dong)(dong)，再放室溫(wen)解(jie)(jie)(jie)凍(dong)(dong)(dong)(dong)(dong)樣(yang)(yang)品(pin)(pin)，反(fan)復(fu)凍(dong)(dong)(dong)(dong)(dong)融(rong) 3 次(ci)，使(shi)細(xi)(xi)(xi)胞(bao)(bao)充(chong)分(fen)(fen)裂(lie)解(jie)(jie)(jie),也可(ke)(ke)(ke)(ke)(ke)將(jiang)(jiang)樣(yang)(yang)品(pin)(pin)進(jin)行超聲破碎，以達到裂(lie)解(jie)(jie)(jie)的(de)目的(de)。4℃ 10000×g 離心(xin)(xin) 10min，除(chu)去細(xi)(xi)(xi)胞(bao)(bao)碎片，取(qu)(qu)上(shang)(shang)清(qing)即(ji)可(ke)(ke)(ke)(ke)(ke)檢測(ce)(ce)，或(huo)(huo)(huo)將(jiang)(jiang)上(shang)(shang)清(qing)置(zhi)(zhi)(zhi)于(yu)-20℃或(huo)(huo)(huo)-80℃保(bao)存(cun)(cun)(cun)，但(dan)應(ying)避免反(fan)復(fu)凍(dong)(dong)(dong)(dong)(dong)融(rong)。細(xi)(xi)(xi)胞(bao)(bao)培(pei)養物(wu)上(shang)(shang)清(qing)或(huo)(huo)(huo)其它生(sheng)物(wu)標(biao)(biao)本：請1000×g離心(xin)(xin)20分(fen)(fen)鐘(zhong)(zhong)(zhong)，取(qu)(qu)上(shang)(shang)清(qing)即(ji)可(ke)(ke)(ke)(ke)(ke)檢測(ce)(ce)，或(huo)(huo)(huo)將(jiang)(jiang)上(shang)(shang)清(qing)置(zhi)(zhi)(zhi)于(yu)-20℃或(huo)(huo)(huo)-80℃保(bao)存(cun)(cun)(cun)，但(dan)應(ying)避免反(fan)復(fu)凍(dong)(dong)(dong)(dong)(dong)融(rong)。.樣(yang)(yang)本保(bao)存(cun)(cun)(cun)和穩定性樣(yang)(yang)本在 2-8℃條件下，可(ke)(ke)(ke)(ke)(ke)以儲(chu)存(cun)(cun)(cun) 72h，在- 20℃或(huo)(huo)(huo)-80℃可(ke)(ke)(ke)(ke)(ke)以儲(chu)存(cun)(cun)(cun)6 個(ge)月及(ji)以上(shang)(shang)。樣(yang)(yang)本收集(ji)后(hou)，不是一次(ci)檢測(ce)(ce)完，請按(an)一次(ci)用(yong)(yong)(yong)量(liang)(liang)分(fen)(fen)裝凍(dong)(dong)(dong)(dong)(dong)存(cun)(cun)(cun)，避免反(fan)復(fu)凍(dong)(dong)(dong)(dong)(dong)融(rong)