DNA Analysis By Flow Cytometry

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DNA Analysis By Flow Cytometry

• ????????????

2
??????

• ?????????46????,???7.10-12 pg DNA/???,??????????,?
  ????????????DNA????????(G0,G1,S,G2
  ,M)?????,DNA?????????????????G1?,????RNA???????,?
  DNA????????

3

• ??S??,DNA????,??????DNA?????G1?G2????DNA??????4??
  ?,????G2?,G2???????RNA????,????M?,??,???DNA??????G
  2??M?????????,??????????,?????????????????,??????
  ?(G0?),?G0??DNA????????G1??????,???????????G0/G1,S
  ,G2/M??

4

• ????????DNA,???????????,???????????????,???G0/G1,
  S?G2/M/,?????????,?????????,???S????????????????
  ?????????????DNA??,???????????/???????????????????
  ?????DNA???????????,?????????????,????????????????
  ???????????

5
??DNA?????
G0/G1????????
G2/M????????
S???
6
????

• ?????????????????????????????????????DNA??????????
  ???????????/??????????????????????????????????????
  ??????????????????????,??????????????????????????

7
??????????

• ???????????????????????????????????????
• ?????????EDTA?Sodium citrate??Ca2?Mg2????,?????
  ?????Triton X-100?NP-40???????????????,????????
• ???????????????,?????????????????????????????????
  ?????

8
?????????

• ????????????106/ ml?????,?????????????,?????????CV
  ?????,????????????,????????,????CV??????
• ???????????????????????????????????????????(???,
  ?????)???????????????S????????????20?

9
??

• ??70???????????????????????????40C????4????
• ???????????,????????PI????????????????????,???????
  ??????????? ??????,??????????????

10
??

• ????????????????488nm??????PI(????),??PI????RNA??,
  ?????????Rnase????????????,????????PI????????20ug/
  106 ??????????50ug/106 ???????lt200C?????30??,3????
  ????

11
?????????

1. ??????200C?,????????
2. PHPI?7.27.6,??????????????????
3. ??????????,??????????,???????????
4. ????????????????????????,???????????
5. ??????????????????,?????????

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DNA?????

• ?????????????????????????????????????????,????????
  ??????????????????????????????(???????)???????????
  ????????????????????????????????????,????????

13
DNA??

• (1)DNA???????????????????(????????????????????????
  ??????????????)?
• (2)???????????????????????CV?????????,?CV?lt2?
• (3)DNA???????????512???????????G1??????PMT???G1??
  ??????????????,?1024???200,512???100?

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• (4)?????DNA?????FL3 Peak 30-40?
• (5)??????????,??????G1????1/10???????
• (6)?????????DNA????????10000??(????)?DNA??????,???
  ?????????????S???,?S???????100????
• (7)?????CV,????????(??100-300???/?)?

15
?????

• ??????FSC vs SSC ????????????????,??????????????,?
  ??????????????
• ????????????????????????????????cytoketatin???????
  ????????????,????????????????CD45???????????????
  ???????????????
• ??Ki67?PCNA???G0?G1??G2?M??

16
????

• Coefficient of Variation(CV)???????????DNA???????
  ????????????
• DNA index(DI)DNA???DI???????G0/G1???????????????G
  0/G1??????????????
• Ploidy????????????????????DNA??????????????DNA???
  ?????????

17
????

• ????
• DI0.91.1 ???
• ???????(DI0.850.9?1.11.15?????DIgt1.15?????DIlt
  0.85??????)
• ???????G2/M????,???G2/M?G0/G1?20????????

18
??????????????

• 1.??DNA??????????????
• 2.?????????,?????????????15?S???,???G0/G1?CV???9
  ????????
• 3.?????????,? G0/G1?CV???,?????1015?S??????????
  ?,???????

19
????

• ????????????
• (1)DNA??,???????DI
• (2)??G1??CV
• (3)S???
• (4)????????????????????CV????