FlowJo FlowJo7.6.5 2012/08/21 FlowJo...1 FlowJo...1 FlowJo (Windows)...1 FlowJo...5 FlowJo...6...6...10 FlowJo...13...13...16...16 FlowJo...18 FlowJo...18 FlowJo...18...24 FlowJo...27...27...32...35 FlowJo...43...43 FlowJo...44 I
...45 FlowJo...47...47 FlowJo...48...51...53 FlowJo...54 CFSE...54 FlowJo...54 FlowJo...58 FlowJo...59 II
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FlowJo FlowJo Leonard Herzenberg FACS 90 FlowJo FlowJo FlowJo 1. PC Windows XP Vista Windows7 512MB 2. OSX10.3 512MB FlowJo (Windows) http://www.flowjochina.com/content/download 1
V7.6.5 http://www.flowjochina.com/sites/default/files/7.6.5_x32.exe FlowJo FlowJo NEXT 2
NEXT NEXT Install Done 3
a. Dongle FlowJo b. Serial Number Use Serial Number c. Get Trial License FlowJo Use Serial Number 4
d. Demo FlowJo Run in Demo Mode Demo FlowJo Demo FlowJo FlowJo 5
FlowJo, 3 color comp Cy5PE comp.fcs 1. FlowJo 2. 3 color comp FlowJo 6
3 color comp 3 color comp Cy5PE comp.fcs 7
1) X SSC SSC 2) Y FSC FSC 3) : 8
1) X Cy5PE:CD4 Y Histogram 2) a. CD4 CD4 9
b. CD4 CD a. WORD PPT b. SVG PNG EMF JPG 10
c. FlowJo d. 11
e. data001.jpg JPG, PNG, GIF, EMF, SVG, PDF FlowJo PNG f. 12
FlowJo FlowJo FlowJo 3-color-experiment 931115- B02-Sample01.FCS 1. 3-color-experiment FlowJo 2. 931115-B02-Sample01.FCS X FSC Y SSC 13
3. X Fluor::CD3 Y FSC CD3 T 14
4. T X Cy5PE::CD4 Y PhyEry::CD8 Q1 CD8 CD8+ T Q2 Q3 CD4 CD4+ T Q4 15
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TREG CD4/CD25/FOXP3 SSC vs. CD4 CD4+ CD25 vs. FOXP3 CD25+/FOXP3+ CD4+/CD25+/FOXP3+ TREG 17
FlowJo FlowJo FlowJo 1. 2. 3. 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
2. Sample Flour FITC comp.fcs 19
3. 2 3 FlowJo 4. > 20
(poor control ) FlowJo 1. 1,2,3 FlowJo PE 21
1) PE cw_size 2) Clear 2. 1) PE FSC/SSC 22
2) X PhyEry::aCD8 Y Histogram PE PE a. b. 3) PE- PE PE+ 23
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
2. X Y T 3. T Change transform values 25
X -10-30 a. b. F 26
FlowJo Titration Data CD8 Sample 1 Sample 2 ~ Sample 8 2 1. Sample 1 27
X FL1-H Y Histogram Sample 1 FL1-H+ Rename CD8+ FL1-H- CD8-2. 28
CD8+ Median Parameter FL1-H CD8 FL1-H 29
Σ(FL1-H):Median CD8- CD8 FL1-H 3. Sample 1 Sample 2 Sample 2 Sample 1 30
Sample 1 Titration Data Sample 1 ALT Sample 1 Titration Data Sample 1 31
1. Sample 1 CD8+ CD8- Σ(FL1-H):Median 32
2. Keyword Antibody Concentration OK 3. CD8+ (FL1-H) Median CD8- (FL1-H) Median CD8 CD8 MFI OK 33
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4. MFI 1 MFI 1. 1. Sample 1 35
FlowJo Titration Data FlowJo 36
2. (Overlay) 1 Titration Data (1) Sample 1 Sample 2 Sample 1 (2) Specify Control Sample 1 Sample 1 OK 37
(3) 38
2 Titration Data (1) Sample 1 CTRL Sample 1 Sample 1 39
(2) 40
(3) (4) coloring filled (5) 41
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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
FlowJo apoptosis ZL-2011-12-20_400.fcs 1. FlowJo X FSC-A,Y SSC-A 2. SSC/FSC 44
X FITC, AnnexinV-FITC Y PE-Texas, PI 1. 2. Q1 (AnnexinV-FITC)-/PI+ Q2: (AnnexinV+FITC)+/PI+ Q3 (AnnexinV-FITC)+/PI- Q4 (AnnexinV-FITC)-/PI- 45
Q2+Q3 AnnexinV 46
FlowJo G1 S G2 M G1 S DNA G2 DNA M DNA PI PI DNA RNA DNA DNA 47
FlowJo cell cycle 5206.001 PI 1. 5206.001 X FSC-H Y SSC-H 48
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
3. FlowJo G1 S G2 G1 CV G2 CV 50
http://www.flowjochina.com/content/tutorial-video#cellcycle 1. RMS RMS(root mean square error) RMS 3.85 2. Watson Dean-Jett-Fox RMS RMS Could not fit. Watson Dean-Jett-Fox 51
3. G1 G2 mean CV 1 G2 G1 2 G1 G2 G1 2 (2G1) G2 G1 G2 0.5 2 G1 G2 G1 mean G1 mean 3 CV DNA G1 G2 CV G1 CV =G2 CV G2 CV G1 CV CV CV CV 52
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
FlowJo CFSE CFSE(CFDA-SE) CFSE CFSE, Proliferation tutorial efluor 670 FlowJo 1. Sample 1 54
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 0.5 CV Background 4. Option 4.1 + 1 1 5 6 #Peaks 6 4.2 Peak 0 unstim #Peaks 1 56
Peak 0 37600 Sample 1 Fix Peak 0 37600 4.3 Ratio Fixed Ratio RMS RMS Ratio 0.5 Peak 0 Ratio CV Background 3 57
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
FlowJo 2 % Divided the percentage of divided cells Div. Index average number of cell divisions % Divided Div. Index 59
Jason J. Coughlin. 2005. RasGRP1 and RasGRP3 Regulate B Cell Proliferation by Facilitating B Cell Receptor-Ras Signaling. The Journal of Immunology 60