Microsoft Word - FlowJo中文实用手册0821.docx

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1 FlowJo FlowJo /08/21 FlowJo...1 FlowJo...1 FlowJo (Windows)...1 FlowJo...5 FlowJo FlowJo FlowJo...18 FlowJo...18 FlowJo FlowJo FlowJo FlowJo...44 I

2 ...45 FlowJo FlowJo FlowJo...54 CFSE...54 FlowJo...54 FlowJo...58 FlowJo...59 II

3 III

4 FlowJo FlowJo Leonard Herzenberg FACS 90 FlowJo FlowJo FlowJo 1. PC Windows XP Vista Windows7 512MB 2. OSX MB FlowJo (Windows) 1

5 V FlowJo FlowJo NEXT 2

6 NEXT NEXT Install Done 3

7 a. Dongle FlowJo b. Serial Number Use Serial Number c. Get Trial License FlowJo Use Serial Number 4

8 d. Demo FlowJo Run in Demo Mode Demo FlowJo Demo FlowJo FlowJo 5

9 FlowJo, 3 color comp Cy5PE comp.fcs 1. FlowJo 2. 3 color comp FlowJo 6

10 3 color comp 3 color comp Cy5PE comp.fcs 7

11 1) X SSC SSC 2) Y FSC FSC 3) : 8

12 1) X Cy5PE:CD4 Y Histogram 2) a. CD4 CD4 9

13 b. CD4 CD a. WORD PPT b. SVG PNG EMF JPG 10

14 c. FlowJo d. 11

15 e. data001.jpg JPG, PNG, GIF, EMF, SVG, PDF FlowJo PNG f. 12

16 FlowJo FlowJo FlowJo 3-color-experiment B02-Sample01.FCS 1. 3-color-experiment FlowJo B02-Sample01.FCS X FSC Y SSC 13

17 3. X Fluor::CD3 Y FSC CD3 T 14

18 4. T X Cy5PE::CD4 Y PhyEry::CD8 Q1 CD8 CD8+ T Q2 Q3 CD4 CD4+ T Q4 15

19 16

20 TREG CD4/CD25/FOXP3 SSC vs. CD4 CD4+ CD25 vs. FOXP3 CD25+/FOXP3+ CD4+/CD25+/FOXP3+ TREG 17

21 FlowJo FlowJo FlowJo FlowJo FlowJo FlowJo 4 1. FlowJo 3 color comp Cy5PE comp.fcs, FITC comp.fcs, PE comp.fcs 3-COLOR.FCS Cy5PE, FITC, PE 18

22 2. Sample Flour FITC comp.fcs 19

23 FlowJo 4. > 20

24 (poor control ) FlowJo 1. 1,2,3 FlowJo PE 21

25 1) PE cw_size 2) Clear 2. 1) PE FSC/SSC 22

26 2) X PhyEry::aCD8 Y Histogram PE PE a. b. 3) PE- PE PE+ 23

27 3. FlowJo 3-color comp > 0 Log 0 Logical FlowJo 3-color comp 3- COLOR.FCS 1. 3-COLOR X CD3 Y CD8 gate1 35.1% 24

28 2. X Y T 3. T Change transform values 25

29 X a. b. F 26

30 FlowJo Titration Data CD8 Sample 1 Sample 2 ~ Sample Sample 1 27

31 X FL1-H Y Histogram Sample 1 FL1-H+ Rename CD8+ FL1-H- CD

32 CD8+ Median Parameter FL1-H CD8 FL1-H 29

33 Σ(FL1-H):Median CD8- CD8 FL1-H 3. Sample 1 Sample 2 Sample 2 Sample 1 30

34 Sample 1 Titration Data Sample 1 ALT Sample 1 Titration Data Sample 1 31

35 1. Sample 1 CD8+ CD8- Σ(FL1-H):Median 32

36 2. Keyword Antibody Concentration OK 3. CD8+ (FL1-H) Median CD8- (FL1-H) Median CD8 CD8 MFI OK 33

37 34

38 4. MFI 1 MFI Sample 1 35

39 FlowJo Titration Data FlowJo 36

40 2. (Overlay) 1 Titration Data (1) Sample 1 Sample 2 Sample 1 (2) Specify Control Sample 1 Sample 1 OK 37

41 (3) 38

42 2 Titration Data (1) Sample 1 CTRL Sample 1 Sample 1 39

43 (2) 40

44 (3) (4) coloring filled (5) 41

45 42

46 FlowJo Annexin V-FITC/PI Annexin V-FITC/PI PS V Annexin V-FITC PS PS PI PS Annexin PI Annexin V-FITC/PI Annexin Annexin, PI 43

47 FlowJo apoptosis ZL _400.fcs 1. FlowJo X FSC-A,Y SSC-A 2. SSC/FSC 44

X FITC, AnnexinV-FITC Y PE-Texas, PI 1. 2.

48 X FITC, AnnexinV-FITC Y PE-Texas, PI Q1 (AnnexinV-FITC)-/PI+ Q2: (AnnexinV+FITC)+/PI+ Q3 (AnnexinV-FITC)+/PI- Q4 (AnnexinV-FITC)-/PI- 45

49 Q2+Q3 AnnexinV 46

50 FlowJo G1 S G2 M G1 S DNA G2 DNA M DNA PI PI DNA RNA DNA DNA 47

51 FlowJo cell cycle PI X FSC-H Y SSC-H 48

52 2. X FL2-W(width) Y FL2-A(area) BD FL2-A FL2-W FL2-A FL2H Beckman X FL3-LIN, PI Y FL3-PEAK, PI 49

53 3. FlowJo G1 S G2 G1 CV G2 CV 50

http://www.flowjochina.com/content/tutorial-video#cellcycle 1.

54 1. RMS RMS(root mean square error) RMS Watson Dean-Jett-Fox RMS RMS Could not fit. Watson Dean-Jett-Fox 51

55 3. G1 G2 mean CV 1 G2 G1 2 G1 G2 G1 2 (2G1) G2 G1 G G1 G2 G1 mean G1 mean 3 CV DNA G1 G2 CV G1 CV =G2 CV G2 CV G1 CV CV CV CV 52

56 1. RMS root mean square error RMS 2. G1 G1 S S G2 G2 3. G1 G1 G2 G2 4. CV coefficient of variation G0/G1 CV CV G1 cv G1 G1 G2 cv G2 53

57 FlowJo CFSE CFSE(CFDA-SE) CFSE CFSE, Proliferation tutorial efluor 670 FlowJo 1. Sample 1 54

58 2. APC_A::670Dye RMS: root mean square error RMS 3. Options 55

#Peaks 8 CFSE 2 CFSE 1/128 efluor 6 8 Peak 0 generation 0 Ratio 0.5 0.

59 #Peaks 8 CFSE 2 CFSE 1/128 efluor 6 8 Peak 0 generation 0 Ratio CV Background 4. Option #Peaks Peak 0 unstim #Peaks 1 56

Peak 0 37600 Sample 1 Fix Peak 0 37600 4.

60 Peak Sample 1 Fix Peak Ratio Fixed Ratio RMS RMS Ratio 0.5 Peak 0 Ratio CV Background 3 57

61 4.4 CV Fixed CV CV 4.5 Background Fixed Backgd FlowJo RMS root mean square error RMS #Peaks: Peak cv CV Peak ratio Undiv. Mean Peak 0 Div. Index Division Index DI Prol. Index Proliferation Index PI % Divided Background Generation 0 Generation 1 58

62 FlowJo 2 % Divided the percentage of divided cells Div. Index average number of cell divisions % Divided Div. Index 59

63 Jason J. Coughlin RasGRP1 and RasGRP3 Regulate B Cell Proliferation by Facilitating B Cell Receptor-Ras Signaling. The Journal of Immunology 60

WinMDI 28

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