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???????,???????????????????,????а???
MTT??????????????л?????3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide, MTT?????????????MTT????????????????????????????????????????????????е?????????????????????????????C????????tetrazolium????????????????formazan????formazan????????????????????????????????????????????????????????MTT???????????????formazan???????50%??N,N-?????????????20%???????????????pH 4.7????MTT???????????????????490 nm????????OD??????????????????????????DMSO?????
MTT??????????????????????????????????????????????????????????????????????????????á?
MTT?????????
1????????????ú?10???С?????????????????????????????
1000??10000??????????96??壬??????200ul.
2:???????????????????????????3??5??????????????????????????????
3???????????3??5???????MTT?????5mg/ml??PBS ??)20ul.
????????4С????????????С?????????????????????????????????????????????????????????????????150ul DMSO????10???????????????
4??????????490nm?????????????????????????????????????????????????????????????????????????????????
???????
??1?????????????????????
??2?????????????????С??10??????????????????????顣??????????????????????????
??3??????????????????в?????????????????????????????鲽?豣??????????????????
MTT??????????????0-0.7????????????Χ????????????
IC50?????????????????????50???????????????????????????????????????????????????????????????????????????????????????50??????????????????????IC50???????2??????????????????????????
???????????????0.1??0.01??0.001??0.0001??0.00001??0.000001?????????10?????????0.1?????????0.95?? 0.80??0.65??0.43??0.21??0.06??????
???????
Pm=0.95
Pn=0.06
P=0.95+0.80+0.65+0.43+0.21+0.06=3.1
Xm=lg0.1=-1
lgI=lg0.1/0.01=1
lgIC50=-1-1*(3.1-(3-0.95-0.06)/4)=-3.6025
IC50=0.00025
????????????ο?;
lgIC50=Xm-I(P-(3-Pm-Pn)/4)
Xm:lg ??????
I:lg(??????/???????)
P:???????????
Pm:???????????
Pn:??С????????
??????=1-?????OD?/??????OD?
????е??????С????????????????С??????
????
??96???????SMMC-7721?????MTT?????????,??ü????1640??????,????MTT??DMSO?????????????????200ul1640,20ulMTT,150ulDMSO ??DMSO?????????????????????DMSO?????H???????б????
??????4000???????????????????20000??/ml??MTT??20ul????????С??????????????????????????????????150ul DMSO??????????????10????????????????
????????IC50????????????????????????????????????????????????1/2-1/3??IC50??????????36h???????????????????????????????1%???????????5-10%??Annexin V??????????????G0????????.
MTT??????Щ???????
(?)???
1.????????????????????????????96???????????????????????????105?????????????????????????????????????????MTT?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????MTT???γ?????????????? ???????????????????????????????????????????
2.????????趨??????????????ο???????????????????Χ??????????????????????С???????Χ???????м??????????????????????????????????Ч???????
3. ??????趨????????????OD???????excel???????????????????????仯??????????仯??????????????????????????????????????????????????????????????????????????????????????
4.???????200ul???????????10??4??5?η???????????????????????68h????????????????????????????????????G0??????????????????????????48h??????
5.MTT??????????????????????????????????????????MTT??????????????????????????????MTT???OD????????
6.????δ??????????????????????????????
7.????????????????????????????????????????MTT???????????????????????????????????????MTT??????????????????????????????????????????????????????
8.????????????ú?15?????????????????????????????????????????????????????????鱾????????????????????????????С??10????????????????С????????????????????????????????
(??)??鲽??
?????????
1.??????????????????????????????????100ul,??????????????????1000-10000???????????????PBS?????
2.5%CO2??37????????????????????????96?????壩,??????????????,???????????????????????С??????????????????????????????壬??????????.???5-7?????,???100ul,??3-5??????.??????5????????????????????
3.5%CO2??37?????16-48С????????????????
4.??????20ulMTT?????5mg/ml????0.5%MTT????????????4h?????????MTT???????????????????????????С????PBS??2-3????????MTT?????????
5.?????????С????????????????
6.??????150ul???????????????????????10min?????????????????????????OD490nm??????????????????
7.???????????????????MTT??????????????????????????????????????????????????MTT?????????????
?????????
1.???????????????????????????1??106/ml?????????????1640??????壩??????40ul ?????Actinomycin D???ж????10ul?????????? l?g/ml??????????????????1:10-1:20)??????????10ul??????????50ul????5??104cell/???????100ul????96??壨???????????????????????????100?(?????100 1640????
2.??37?棬5%CO2????16-48С????????????????
3.??????10 ul MTT?????5 mg/ml????0.5%MTT????????????4 h?????????????????WST-1??????4 h???????????4??????????????????OD570nm??630nmУ???????????????????
4.?????1000?x10min????С?????????壬??????100 ul???????????????????????10 min?????????????????????????OD570nm??630nmУ???????????????????
5.???????????????????MTT??????????????????????????????????????????????????MTT???????????????????趨3?????
(??)MTT??????
MTT???????????????????4oC?????????????Ч?????????5mg/ml??????-20???????棬??????????????С??????????????????????????????????????????????MTT??????EP??????????????????????????м??????????????????,????MTT????????????????????????
MTT???°??????????С??,?????????????????????????.????MTT????????MTT????????У???96?????????????й????????????????????????????????????????????????
????MTT???PBS???,????????????????60?????????
PBS??:
Nacl 8g Kcl 0.2g Na2HPO4 1.44g KH2PO4 0.24g ??ph 7.4 ????1L
(??)????????????(???)
???????30????????????,???????????;????????ú?????????壩??????????????????????????顣
?????????????????????????????, ???????????10000/ml?????????????OD??????伴????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????MTT??????????10000/ml??100ul/???
?????????????????????????.???????????????????д??????????С???????????????????????????????????????????????????????????????????????????????á????????????????????105,??????????103-104. |
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