Flow Cytometry

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• ?????
• Flow Cytometry

(FCM)
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????????
????????
BD FACSVantage
BD-Calibur
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??  ??

• 1. ????
• 1934 Moldavan
• 1973 Steinkamp

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• 2. ?????????????????????????????????????.
• 3. ??
• ?????,???????????????
• ????????
• ?????????????????????,??????

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4. ????

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• ???????,???????????,???????????????????????????,?
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????????,???????????????? ???????????????,?????
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(No Transcript)
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• ??????(FSC)???????????
• ??????(SSC)??????????????????

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• ????

• ????(FL)????????????????????,???????????????
• ???????????????????(??)
• ?FL????FL??????(??)
• ?????????????????(488nm),?????FL,?FL1?FL2?FL3,????
  ???????????????(488nm?633nm?325nm),???6?FL?

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• ?????????

??????????????? ????
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? ?????????
???
FSC SSC FL1 FL2 FL3
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?? ?? ?? ?? ??
????,????
(??,??,??)
????,????
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• ????????????,?????????????????????????????????
• ????
• Gated???????????????????????
• Mean??????,????????????,????????????
• Geo Mean????????,?????????????????

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? ?????????????
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? Annexin V/PI????????????
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• ????

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• ?????

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??100101(M1)?????,101???(M2)?????
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• ????/??/???????
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• ???/??????
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• pH?
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• ????????????

• ????
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• ?????
• ????
• DNA???????
• RNA??
• ?????

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• ?) ??DNA????

??????PI (Propidium iodide????),??,??PI????????DNA
,?????PI????????????,??????,??????DNA?????????????
?????????
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(No Transcript)
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• ??????

??2N??G0/G1???,4N??G2/M???,??????S???????2???4????
?????DNA?
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DNA??????

DNA??
M
G0
G2
G1
????
s
s
????
DNA??
200
400
600
800
1000
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(No Transcript)
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????????DNA??
2??
???
4??
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???????

• DNA?G1??????PI??,?????G1?DNA???????,????????
• ?????,?????????,?DNA??,???????,????????????,??????
  ????,???????DNA????????,????DNA????,???2???

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(No Transcript)
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• ?)????????????

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1. ????

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

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????????DNA??
2??
???
4??
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(No Transcript)
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2. ????

• ?G0/G1???????????,?????
• ????,???????????????????????????

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(No Transcript)
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Annexin V Assay
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? Annexin V/PI????????????
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Date acquired 14-Jan-03 File 7Gy
4hr.001 Source dongbo DIPLOID 100.00 Dip
G0-G1 73.12 at 48.16 Dip G2-M 9.59 at
96.33 Dip S 17.29 G2/G1 2.00 Dip CV
6.04 Apoptosis 13.66 Mean 27.48
? FCM????? ??????
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????????????

• ????,???????,????????????????????????????????????
  ????????,???????????????
• ?????FSC ?SSC ?
• ?????,????,FSC ?,SSC ?

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?)??????

• CD3(T??,CD4?CD8))?CD19(B??)?CD16?CD56(NK??
  ),????????????????????????????????????????????????
  ?

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(No Transcript)
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???????????
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?)????????

• ???????????????????????????????,???????????????,?
  ?????????????????????
• FCM?????????, ????????,????,??????????????????,???
  ?????????????????????????????????

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?) ????????

• ?????????????????????,????????????,???????
  ,???????????????????????????????FITC?

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• ???????,????????????

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??(????)
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• ?)????????
• 1106/ml?????,
• ??????1-5µmol/ml Fluo-3/AM,????,??????60???
• ?HEPES???Hanks????????,???0.5ml?????,?????????
• ??????????????????????????

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FCM???????? ?????????
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M1???????????????!?????????????,????????????????
??,??????????????????????????????????,????????,???
??????????
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?. ??

• ??????????,?????????,
• ????????????????,?????????????300???,
• ?????????????????

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Fluorescence Activated Cell Sorting
FALS Sensor
488 nm laser
Fluorescence detector
-
Charged Plates
Single cells sorted into test tubes
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• ??-----??????????????

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