(Microsoft Word - FACSCanto User Guide\272\353\302\262\252\251.doc)

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1 BD FACSCanto BD FACSCanto 流式細胞儀 488nm 20mw 633nm 17mw % 120uL/min 10,000events/sec BD FACSCanto 1

2 BD FACSCCanto BD FACStation 1.1 FACSCanto BD FACSCanto 2

3 1.1.1 Fluidics Sample injection tube Aspirator arm flow cell SIT SIT Port or Switch flow cell tubing SIT Sample Injection Tube adapter lever aspirator arm aspirator arm bar SIT BD FACSCanto 3

4 Flow Cell Optics FACSCanto BD FACSCanto 488nm 633nm nm mw Coherent R Sapphire TM FITC, PE, PE-Texas Red, PerCP, PerCP-Cy5.5, PE-Cy7,PI JDS Uniphase TM HeNe APC,APC-Cy7 BD FACSCanto 4

5 BD FACSCanto 5

PMT LP Filter BP or LP Filters Fluorochromes Position nm nm 488 Laser Blue A: 735 780/60 PE-Cy7 Blue B: 655 670LP PerCP, PerCP-Cy5.

6 PMT LP Filter BP or LP Filters Fluorochromes Position nm nm 488 Laser Blue A: /60 PE-Cy7 Blue B: LP PerCP, PerCP-Cy5.5 Blue C: /42 PE Blue D: /30 FITC Blue E: Blank filter 488/10 SSC 633 Laser Red A: /60 APC-Cy7 Red B: Blank filter 660/20 APC Port or Button a. System Power FACSCanto b. Power Out FACSCanto c. Air In d. Waste (A) e. Communications f. Sheath (B) g. Waste (A) BD FACSCanto 6

7 psi FACSFlow 10 5 FACS Shutdown 5 BD FACSCanto 7

8 2.4 h. On/Off FACSCanto Off i. Fluid Out j. Communication k. Power In FACSCanto l. Auxiliary air in m. Air Out n. Waste o. Waste BD FACSCanto 8

9 ~70psi 50psi BD (0800-SERVIC) BD FACSCanto 9

10 1.3 BD FCASCanto BD FACSCanto FACSCanto BD FACSCanto Clinical or FACSDiva BD PC Pentium R 1GB RAM 40GB Windows R XP(US-English) 17 BD FACSCanto Clinical Software Instrument QC, Lymphocyte subset TBNK, HLA-B27, Stem Cell Enumeration BD FACSDiva BD FACSDiva Data Manager Windows MS Office 2003 BD FACSCanto User s Guide BD FACSCanto 10

11 BD FACSCanto BDISEnter 3. BD FACSDiva 4. Instrument Instrument Disconnected FACSDiva 5. Instrument Instrument Connected Instrument FACSFlow 6. Shutdown solution Sheath buffer System Prime -- Bubble filter purge Degas flow cell 5~10 BD FACSCanto 11

12 Instrument > Automatic Clean FACSDiva FACSDiva Automatic Clean Automatic Clean 7. OK 8. Instrument Remaining warmup time 7:27 The system is ready 9. BD FACSCanto 12

13 2.2 (Controls in Instrument Frame) Fluid Level Indicators BD FACSDiva FACSFlow 17% 34% 51% 68% shutdown cleaning 20% FACSFlow FACSFlow ( ) ( ) FACSFlow 0% >99% Laser Tab Window Extension FSC Area Scaling BD FACSCanto 13

14 Status Tab 2.3 Acquisition Dashboard BD FACSCanto 14

15 Current Activity Basic Controls Next Tube Remove Tube SIT Acquire Data Record Data list mode data Restart Acquire Record Remove Tube Remove Tube FACSCanto Aspirator arm SIT Acquisition Controls Remove tube Aspirator arm Aspirator arm SIT BD FACSCanto 15

16 Carousel Controls (Run) (Run Single Tube) (Mix) (Skip) (Rerun) (Paus e) Acquisition Setup 10, (Events to Display) (Flow Rate) Low 10 µl /min Medium 60 µl /min High: 120 µl /min BD FACSCanto 16

2.4 1. Automatic Clean 2. 55~65 Psi 3. 1X10 6 cells/ml 0.5 ml 4. FALCON 2052 5. 6. 7. 8. 2.5 Fluidics Shutdown BD FACS Shutdown solution 1. 3 ml FACSClean 10%Bleach 2.

17 Automatic Clean 2. 55~65 Psi 3. 1X10 6 cells/ml 0.5 ml 4. FALCON Fluidics Shutdown BD FACS Shutdown solution 1. 3 ml FACSClean 10%Bleach 2. Basic Controls Acquire Data 3. Medium Flow Rate Acquire 5 5 Acquire 4. FACSClean 3 ml dh2o 5. Basic Controls Acquire Data Medium Flow Rate Acquire Acquire Data Fluidics Shutdown BD FACSCanto 17

7. File > > Quit Fluidic Shutdown 8. OK FACScanto Fluidics Shutdown FACSDiva 1 ml SIT 9. 1/10 2.

18 7. File > > Quit Fluidic Shutdown 8. OK FACScanto Fluidics Shutdown FACSDiva 1 ml SIT 9. 1/ FACSCanto FACSCanto Instrument/Clean Modes/Clean Flow Cell.. 3 ml FACSClean FACSClean fluidics startup fluidics shutdown BD FACSCanto 18

19 CV Instrument > Cleaning Modes > De-gas Flow Cell.. Instrument > Cleaning Modes > Bubble Filter Purge.. Priming Instrument > Cleaning Modes > Prime After Tank Refill.. OK (Long Clean) Long Clean FACSClean FACS shutdown 75 FACSClean >275 ml >1,100 ml Instrument > Cleaning Modes > Long Clean.. 70 BD FACSCanto 19

20 2.7 FACSCanto BD FACS 7-color setup beads Cat. No Rainbow fluorescent particles Cat. No Nile Red fluorescent particle Cat. No BD FACSFlow sheath fluid Cat. No BD FACS cleaning solution Cat. No BD FACS shutdown solution* Cat. No mL polystyrene test tubes (BD Falcon ) uncapped, 125 per bag Cat. No capped, 125 per bag Cat. No capped, 25 per bag Cat. No with cell-strainer cap Cat. No *BD FACS shutdown solution BD FACSCanto 20

21 BD FACSCanto Instrument configuration & User Preferences Experiment Instrument Setting ( ) Instrument Setting ( ) BD FACSCanto 21

22 3.1 Verify instrument configuration and user preferences 1 2 BD FACSDivia 3 Edit>User Preferences General OK 4 2-laser, 6-color Instrument>Instrument Configuration Duplicate Set OK BD FACSCanto 22

23 5 FITC PE PerCP PE-Cy7 APC APC-Cy Browser ( ) Instrument ( ) Inspector ( ) Workseet ( ) Acquisition Controls ( ) 2 Browser New Folder Folder_001 3 Folder_001 Immunophenotype Pop-up Menu Immunophenotype 4 Immunophenotype New Experiment Experiment_001 BD FACSCanto 23

Instr Settings ( ) 8 Inspector Parameters Parameters

24 5 Experiment_001 Pop-up Menu 5-Color Exp 6 Experiment Inspector Use Global Instrument Settings 7 5-Color Exp Instr Settings ( ) 8 Inspector Parameters Parameters FITC PE PerCP APC APC-Cy7 Parameters Add or Delete BD FACSCanto 24

25 9 Instrument>Instument Setup>Creat Compensation Tubes OK Add or Delete Compensation Controls ( ) Experiment Compensation Controls Stained Controls ( ) BD FACSCanto 25

26 3.3 Optimize Settings FSC SSC CD4 FITC CD16+56 PE CD3 PerCP CD19 APC CD8 APC-Cy7 BD FACSCanto 26

27 FSC SSC FSC SSC FSC/ SSC Inspector Parameters 1 Unstained ControlAcquisition Setup Event to Display ( ) 500evt 2 3 Basic Controls Acquire Data Record BD FACSCanto 27

28 4 FSC SSC 5-1 Instrument Parameters FSC SSC FSC SSC Acquire Data Ctrl 10 FSC 1 Basic Controls Acquire Data 2 Instrument Threshold FSC FSC BD FACSCanto 28

29 1 Unstained Control P1 P1 P1 5-1 FSC SSC 2 P1 Apply to all Compensation Control P1 3 Basic Controls Acquire Data BD FACSCanto 29

30 4 Edit>Select All 5 Inspector ( ) Plot () Show Grid Show Grid Show Grid ( ) , FSC SSC 6 Basic Controls Acquire Data BD FACSCanto 30

7 5-2 Basic Controls Acquire Data 5-2 8 Basic Controls Record Data ( ) 9 Remove Tube( ) 10 Basci

31 7 5-2 Basic Controls Acquire Data Basic Controls Record Data ( ) 9 Remove Tube( ) 10 Basci Controls Next Tube FITC Stained Control Unstained Control Tube Compensation Controls BD FACSCanto 31

32 3.4 1 FITC Basic Controls Acquire Data 2 P1 Basic Controls Record Data 3 Remove Tube 4 Next Tube ( ) Compensation Controls Single Stained Control P2 1 FITC Stained Control BD FACSCanto 32

33 2 FACSDiva P2 5-3 P2 5-3 FITC P2 3 PE Stained Control PE 4 2 P2 5 P2 P2 6 P2 7 Instrument>Instrument Setup>Calculate Compensation 8 (Compensation Matrix) Experiment Instr Settings Experiment+ OK BD FACSCanto 33

34 BD FACSDvia Global Worksheet 4.1 Global Worksheet 1 5-color exp Specimen Pop-up Menu Specimen_001 LWB 2 LWB Tube_001 + Tube_001 Tube _001 T/B/NK_001 3 T/B/NK_001 4 Basic Controls Next Tube Tube Tube T/B/NK_002 Next Tube( ) Tube Tube Tube BD FACSCanto 34

35 Next Tube Tube Tube Next Tube Next Duplicate without Data ( ) 5 Experiment>Experiment Layout Experiment Layout ( ) 6 Lable ( ) Tube CD3 BD FACSCanto 35

Worksheet Dot Plot 10 9 11 1 FSC/SSC 2 FITC/PE 3 PerCP/PE 4 APC/PC-Cy7 4.

36 7 Acquisition ( ) T/B/NK_001 T/B/NK_002 Events to Record ( ) 10,000 OK Inspector Acquisition 8 Normal/Global Worksheets Global Worksheets Normal Workseets ( ) Global Worksheets ( ) 9 Global Worksheet Dot Plot FSC/SSC 2 FITC/PE 3 PerCP/PE 4 APC/PC-Cy CD4 FITC/CD16+CD56 PE/CD3 PerCP/CD19 APC/CD8 APC-Cy7 2 T/B/NK_001 3 Basic Controls ( ) Acquire Data 4 FSC/SSC P1 5 Shift BD FACSCanto 36

Controls Next Tube 10 Acquire Data 11 P1 Basic Controls

37 6 Show Population Show P1 P1 7 populations marker quadrants 8 Basic Controls Record Data 10,000 9 Basic Controls Next Tube 10 Acquire Data 11 P1 Basic Controls Record Data 12 Experiment Instr Settings Inpector Print BD FACSCanto 37

38 Typical Instrument Setting for 6 Color BD FACSCanto 38

4.3 5-4 BD FACSDiva Analysis 1 New Global Sheet ( ) Global Sheet2 (

39 BD FACSDiva Analysis 1 New Global Sheet ( ) Global Sheet2 ( ) 2 Global Sheet2 ( ) T/B/NK Analysis 3 Dot Plot ( ) BD FACSCanto 39

4 T/B/NK_001 FSC SSC CD3 PerCP CD16+56 PE CD3 PerCP CD19 APC CD3 PerCP CD8 APC-Cy7 CD3 PerCP CD4 FITC 5 5 4 1 T/B/NK_001 Tube ( ) T/B/NK_001

40 4 T/B/NK_001 FSC SSC CD3 PerCP CD16+56 PE CD3 PerCP CD19 APC CD3 PerCP CD8 APC-Cy7 CD3 PerCP CD4 FITC T/B/NK_001 Tube ( ) T/B/NK_001 T/B/NK Analysis 2 P1 3 Show Population Hierarchy 4 Population Hierarchy lymphocyte 5 Shift FSC/SSC Show Population lymphocyte BD FACSCanto 40

41 6 Shift Inspector Title Title Tube Population Title Tube Population 7 CD3 PerCP CD16+56PE CD3 T Cell 8 CD16+56 NK Cell 9 CD3 PerCP-Cy5.5/CD19 APC CD19 B Cell 10 T Cell CD3 PerCP/CD8 APC-Cy7 Cytotoxic T Cell T Cell Cytotoxic T Cell T Cell Sub-population 11 TCell CD3 PerCP/CD4 FITC Helper T Cell BD FACSCanto 41

42 1 Creat Statistics View 2 Edit Statistics View 3 Edit Statistics View Population # Events ( ) %Parent (% )Population Hierarchy %Parent (% ) 4 Edit Statistics View Statistics lymphocytes Mean ( ) BD FACSCanto 42

43 P1 1 Analysis CSV File/Export/statistics BD FACSCanto 43

44 3 LymphoSubsets Save 4 Basic Controls Next Tube T/B/NK_002 T/B/NK Analysis File/Export/Statistics LymphoSubsets Save Append CSV BD FACSCanto 44

45 4.4 Log Out 1. File/Quit Log Out FACSDiva 2. Quit BD FACSCanto 45

46 (For Stardard FACSCanto) (nm) (nm) Fluorescein, FITC (Blue D) Phycoerythrin-R, PE (Blue C) Peridinin-chlorophyll, PerCP (Blue B) PerCP-Cy (Blue B) Cy-Chrome (Blue B) PE-Texas Red (B or C) PE-Cy (Blue B) PE-Cy (Blue A) Allophycocyanine (Red B) Ethidium Bromide 510 (+ds DNA) 595 (Blue C) APC-Cy (Red A) Propidium Iodide 536 (+ds DNA) 623 Acridine Orange 480 (+ DNA) 520 (Blue D) (+ RNA) 650 (Blue B) Viability Propidium Iodide (B or C) Thiazole Orange 509 (+ RNA) 533 (Blue D) YOPRO (Blue D) Acridine Orange (Blue D) DiO-C6 (3) (Blue D) Mitochondrial ph BCECF-AM 488 Ratio 520/620 SNARF1-AM 514 Ratio 587/640 Fluo4-AM (Blue D) Calcium Green (Blue D) 7-AAD (Blue B) Rhodamine (Blue D) Fura Red (Blue B) H2O2 sensitive Dihydrorhodamine (Blue D) DCFH-DA (Blue D) O2- radical sensitive Hydroethidine (Blue C) Esterase sensitive Fluorescein -DA (Blue D) BD FACSCanto 46

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untitled 2008/02/05 Isoiso FACSCalbur 488nm FL1 530/30 FITC GFP alexa488 FL2 585/42 PE Cy3 PI FL3 650LP Percp PC5 PI PE-C 7 Red APC Falcon BD Calibrite 3 BD No.340486 DNA QC particles BDNo.349523 Flow-Cheak(10um)