采样HAHI洗血配制培养液等步骤

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,采样、HA、HI、洗血、配制培养液等步骤,1,洗血,1ml 肝素可以抗凝 5.5ml血液,心脏抽取豚鼠血液2ml,在离心管里面加PBS至10ml,离心 3000rpm/ 5min,用塑料吸管弃上清，再加PBS至10ml,离心 1,2,离心 2,离心 3000rpm/ 5min,弃上清，加PBS至10ml,离心 4000rpm/ 10min,去上清，用塑料吸管将血细胞吸入玻璃瓶,加PBS稀释到0.6%，4,%,保存备用,3,采样,地点：汕头大学医学院附属第一医院儿科,时间：周一周五 上午9:00 11:00,发烧儿童体温高达37.7以上，在7天内出现疑似病毒感染的呼吸道症状，年龄0-18岁，经就诊儿童父母同意采集鼻咽拭子，采完装入采样管，放入冰包，同时做好记录。,4,采样液配制,以100ml为例：,100ml MEM,10ml PS,1ml 制霉菌素,5,标本稀释液,100ml MEM,5ml PS(青链霉素),6,病毒稀释液,100ml MEM,200ul TPCK胰酶,1ml PS,7,细胞培养液,100ml MEM,10ml FBS,1ml PS,8,种标本,STUC附属第一医院采取流感症状发热儿童咽拭子,咽拭子在振荡器上面振荡，使棉签中的标本充分溶解于液体中,用移液枪取220ul样本稀释液加入到1ml离心管中,吸取275ul标本液加入该离心管中,振荡、在室温作用1-2小时后离心：10000rpm/ 3min,9,用PBS将细胞培养管洗两遍,吸取420ul离心后的标本液加入细胞培养管,35吸附1-2h,加维持液后放入培养箱里，培养5d，每天观察CPE,阳性,阴性,冻存或者继续接种细胞,盲传，同时冻存,10,HA test,红细胞凝集实验,11,1,2,3,4,5,6,7,8,9,10,11,12,A,B,C,D,E,F,G,H,100ul virus,50 50 50 50 50 50 50 50 50 50 50,50 50 50 50 50 50 50 50 50 50 50,step1: add 50ul PBS in 2-12,step2: add 100ul virus in 1,step3: coubling dilution,titer: 1 2 4 8 16 32 64 128 256 512 1024 2048,titer=2,n-1,12,取96孔板，用记号笔做好标记,在212号排列孔里各加入50ml PBS,在1号排列里各加入100ml 病毒液,用排枪从1号孔吸取50ml，加入2号孔，反复吹吸，吸取50ml加入3号管,112号孔里面各加入50ul 火鸡血/豚鼠血,火鸡血用U型板,豚鼠血用V型板,静置30min，判读,13,阳性,阴性,冻存或者继续接种细胞,盲传，同时冻存,吸取500ul 病毒液，离心:10000rpm/3min,将细胞培养管用PBS洗两遍，之后加入病毒液,35吸附1-2h,加维持液，培养5d，每天观察CPE,14,HI test,红细胞凝集抑制试验,15,Adjust 4AD,Then the HA titer,4,titer=2,n-1,titer=4,50ml,X,X=,50ml,2,n-3,X 为第一个孔里面要加的病毒量，然后加PBS至50ml,=,或者公式二,V,总,V,virus,HA,8,16,1,2,3,4,5,6,7,8,9,10,11,12,A,B,C,D,E,F,G,H,H,2,O:50X,Virus: X,17,1,2,3,4,5,6,7,8,9,10,11,12,A,B,C,D,E,F,G,H,50ul serum,25ul PBS,25ul,add 25ul virus to every well,wait for 45min,add 50ul blood to every well,18,Cell culture,Cell culture flask:,S 25cm,2,5ml,M 75cm,2,1015ml,L 175cm,2,30ml,Dilution ratio/dilutionrate:,Two days 1 to 810,Three days 1 to 20,19,Plan,Cell culture tube 1ml,96 well plate 9620ul (1980ul),If you need 200 tubes in 2 days, the plan as follow:,Total need cell culture fluid: 201ml,20ml/10(dilutionrate for 2 days)=2ml(take 2ml from the 810ml of diluted from M flask),20,If you need 20 96-well plates in 2 days, the plan as follow:,Need total volume:209620ul=40ml,Cell culture flask:40ml/10=4ml (take 4ml from the 810ml of diluted from M flask),21,