BD FACSCanto. BD FACSCanto. 488nm 20mw 633nm 17mw 0.1% 120uL/min 10,000events/sec. BD Biosciences 1

Transcription

1 BD FACSCanto BD FACSCanto 488nm 20mw 633nm 17mw % 120uL/min 10,000events/sec BD Biosciences 1

2 BD FACSCCanto BD FACStation 1.1 FACSCanto

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 Biosciences 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

5 BD Biosciences 5

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 Blue F: Optional Optional Blue G: Optional Optional Blue H: Optional Optional 633 Laser Red A: /60 APC-Cy7 Red B: Blank filter 660/20 APC Red C: Optional Optional 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)

7 psi FACSFlow 10 5 FACS Shutdown 5 BD Biosciences 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

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

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

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

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 Biosciences 13

14 Status Tab 2.3 Acquisition Controls Next Remove Tube SIT Acquire Record list mode data Restart Acquire Record Remove Tube Remove Tube FACSCanto Aspirator arm SIT

15 Acquisition Controls Remove tube Aspirator arm Aspirator arm SIT Flow Rate Low 10 µl /min Medium 60 µl /min High: 120 µl /min BD Biosciences 15

16 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. Acquisition controls Acquire 3. Medium Flow Rate Acquire 5 5 Acquire 4. FACSClean 3 ml dh2o 5. Acquisition controls Acquire Medium Flow Rate Acquire Acquire Fluidics Shutdown

17 7. Instrument > Fluidics 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 Biosciences 17

18 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

19 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 Biosciences 19

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

21 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 OK BD Biosciences 21

22 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

23 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 Biosciences 23

24 9 Instrument>Instument Setup>Creat Compensation Tubes OK Add or Delete Compensation Controls ( ) Experiment Compensation Controls Stained Controls ( )

25 3.3 Optimize Settings FSC SSC CD4 FITC CD16+56 PE CD3 PerCP CD19 APC CD8 APC-Cy7 BD Biosciences 25

26 FSC SSC FSC SSC FSC/ SSC Inspector Parameters 1 Unstained ControlAcquisition Controls Event to Display ( ) Acquisition Controls Acquire Record

27 4 FSC SSC 5-1 InstrumentParameters FSC SSC FSC SSC Acquire Ctrl 10 FSC 1 Acquisition Controls Acquire 2 InstrumentThreshold FSC FSC BD Biosciences 27

28 1 Unstained Control P1 P1 P1 5-1 FSC SSC 2 P1 Apply to all Compensation Control P1 3 Acquisition Controls Acquire

29 4 Edit>Select All 5 Inspector ( ) Plot ( ) Show Grid Show Grid Show Grid ( ) , FSC SSC 6 Acquisition Controls Acquire BD Biosciences 29

30 7 5-2 Acquisition Controls Acquire Acquisition Controls Record ( ) 9 Remove Tube( ) 10 Acquisition Controls Next FITC Stained Control Unstained Control Tube Compensation Controls

31 3.4 1 FITC Acquisition Controls Acquire 2 P1 Acquisition Controls Record 3 Remove Tube 4 Next ( ) Compensation Controls Single Stained Control P2 1 FITC Stained Control BD Biosciences 31

32 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

33 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 Acquisition Controls Next Tube Tube T/B/NK_002 Next ( ) Tube Tube Tube Next Tube Tube BD Biosciences 33

34 Tube Next Tube Next Duplicate without Data ( ) 5 Experiment>Experiment Layout Experiment Layout ( ) 6 Lable ( ) Tube CD3

35 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 Acquisition Controls ( ) Acquire 4 FSC/SSC P1 5 Shift BD Biosciences 35

36 6 Show Population Show P1 P1 7 populations marker quadrants 8 Acquisition Controls Record 10,000 9 Acquisition Controls Next ( ) 10 Acquire 11 P1 Acquisition Controls Record 12 Experiment Instr Settings Inpector Print

37 BD Biosciences 37

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

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 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 Biosciences 39

40 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

41 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 Biosciences 41

42 P1 1 Analysis CSV File/Export/statistics

43 3 LymphoSubsets Save 4 Acquisition Controls Next T/B/NK_002 T/B/NK Analysis File/Export/Statistics LymphoSubsets Save Append CSV BD Biosciences 43

44 4.4 Log Out 1. File/Log Out FACSDiva 2. Quit

45 FACSCanto BD Rainbow Fluorescent Particles 3.2µm 488 nm 633 FACSCanto FL1-6 (Mean Channel Number) (CV) Optical Alignment Log Levey-Jennings 1 BD Biosciences 45

46 1 Levey-Jennings Instrument configuration & User Preferences QC 5.1 Verify instrument configuration and user preferences 6 7 BD FACSDiva 8 Edit>User Preferences General OK

47 9 Instrument>Instrument Configuration 2-Laser, 6-Color Set Configuration QC OK 10 FITC PE PerCP PE-Cy7 APC APC-Cy7 5.2 QC 10 Browser New Folder Folder_001 BD Biosciences 47

48 11 Folder_001 Instrument QC Pop-up Menu Instrument QC Enter

49 12 Instrument QC New Experiment Experiment_ Experiment_001 Pop-up Menu Feb 03 Enter 14 Specimen QC_ QC_ Tube_001 + Tube_ Acquisition Template 1. Normal/Global Worksheets Global Worksheets QC 2. Global Worksheet Dot Plot FSC-H vs SSC-A 3. Histogram FSC-A SSC-A FITC-A PE-A BD Biosciences 49

50 PerCP-Cy5.5-A PE-Cy7-A APC-A APC-Cy7-A

51 4. Global Worksheet Create Statistics View 5. Edit Statistics View 6. Edit Statistics View Populations # Events %Parent Population Hierarchy %Parent 7. Statistics (mean) CV CVs 1 OK Figure? BD Biosciences 51

52 BD Falcon ml BD FACS Flow Sheath Buffer 2 9. Tube_ Acquisition Controls Event to Display 500 evt Low Acquisition Controls Acquire Record 13. Instrument Parameters FSC SSC x10 FSC SSC Acquire Ctrl 10

53 14. FSC/SSC P1 15. Inspector P1 P1 16. FITC PMT FITC-H 100x x Acquisition Controls Acquire 19. All compensation set 0% BD Biosciences 53

54 Acquisition Controls Events to record =10,000 evt Record (Interval Gate) 23. Show Population Hierarchy 24. File>Export>Statistis 25. Instr Settings QC 26. QC Log mean CV

55 Sample Quality Control (QC) Log BD Biosciences 55

56 1A OPTICAL ALIGNMENT LOG Particle Lot no. LASER1: Wavelength nm Power mw Current amps LASER2: Wavelength nm Power mw Current amps FSC SSC FL1 FL2 FL3 FL4 FL5 FL6 Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV 56

57 BD FACSDiva BD FACSDiva BD FACSCanto BD FACSDiva 6.1 BD FACSDiva BD FACSDiva ReadMe > Start>Programs>BD FACSDiva Software Program Files Program Files\BD FACSDiva Software ReadMe BD FACSDiva PDF BD FACSDiva Software BD FACSDiva BD Browser ( ) Experiment () Experiment () 57

58 6.2 BD FACSDiva BD FACSDiva 6-1 BD FACSDiva 58 BD FACSCanto

59 59

60 6.3 Browser ( ) BDFACS Experiment () Folder () Experiment () Experiment () 60 BD FACSCanto

61 Experiment () Tube ( ) New Sort Layout ( ) Experiment () Specimen () Tube ( ) Experiment () Experiment () Experiment () Experiment () Search ( ) Experiment () Experiment () Display All ( ) Experiment () Experiment () Experiment () Experiment () Display All ( ) 61

62 6.4.2 Edit>Find Ctrl-F Find ( ) Fluorochrome Label ( ) CD4 Search ( ) Experiment () Specimen ()Tube ( ) 5/17/01 May 17, 2001 Experiment () Experiment () Experiment () Experiment () Acquisition Controls ( ) 62 BD FACSCanto

63 6.4.4 BD FACSDiva Experiment ()

64 64 BD FACSCanto

65 6.5 BD FACSDiva Folder () Experiment () Specimen () Folder () Experiment () Specimen () Edit>Find Experiment () Specimen () / / Tube ( ) Folder () Experiment () Specimen ( )

66 6-6 Acquisition Template ( ) 6 Color / / Experiment () Edit>Find A Experiment () Edit>Find BD FACSCanto

67 6.6 Worksheet BD FACSDiva Normal Worksheets Tube ( ) Tube ( ) Tube ( ) P1 Tube ( ) P1 Tube ( ) Global Worksheets Tube ( ) Tube ( ) Tube ( ) Tube ( ) Tube ( ) Tube ( ) 67

68 6.7 Instrument Configuration PerCP-Cy55 B PerCP B PI B 6-9 PerCP-Cy55 Experiment () BD FACSCanto

69 6.8 Instrument Setup Tube ( ) Specimen ()Experiment () Tube Tube Tube Tube Experiment Tube 69

70 1.1 (fluorophore) FITC phycoerythrin ( ) ; FITC 1.2 bandpass Dichroic bandpass ( FL1 FACSCanto Blue D) FACSCanto 30nm 530/30 515nm 545nm 2 530/30 FITC 2 70 BD FACSCanto

71 1.3 Spectral spillover PE ( FL2 585/42 FACSCanto Blue C) PE FITC FITC ( FL3 585/42 FACSCanto Blue B) FITC FITC PE 4 4 FITC PE FITC 576nm A PE PE FITC 576nm PE PE 530nm B FITC >2 Fluorescence compensation 71

72 FITC PE PE FITC BD FACSCanto

73 nm N 0 FITC PE A Auto/Auto FITC PE B FITC Auto 73

74 PE PE PE FITC PE FITC PE FACScan/FACSCalibur FACSCanto FACSDiva A FITC PE FITC PE FITC 1,000 PE PE 200-1,000x PE 200-1,000x PE 200-1,000x20 =0 4 30PE 200-1,000x30 = 10 PE FITC PE PE FITC PE PE A FITC PE A C Auto PE PE FITC C PE PE FITC FITC 74 BD FACSCanto

75 1.6 FITC PE PerCP PE-Cy7 FITC- PE FITC- PerCP FITC- PE-Cy7 PE- FITC PE- PerCP PE- PE-Cy7 PerCP- FITC PerCP- PE PerCP- PE-Cy7 PE-Cy7- FITC PE-Cy7- FITC PE-Cy7- FITC 30 Compensation Matrix F ACSCanto Full Matrix & Advanced Digital Color C ompensation 6 FACSDiva 75

76 CD4 FITC/CD16+CD56 PE/CD3 PerCP/CD19 APC/CD8 APC-Cy7 T B NK CD4 FITC CD16+CD56 PE CD3 PerCP CD19 APC CD8 APC-Cy7 CD4 FITC T CD16+CD56 PE NK CD3 PerCP T CD19 APC B CD8 APC-Cy7 T 76 BD FACSCanto

77 FACSDiVa BD BD FACSDiVa 18 bit FCS3.0 CellQuest Pro, FCS Express, WinMDI, ModFit FCS2.0 FCS3.0 LT (Export FCS) (Export Experiments) 2.1 FCS FCS FCS2.0 FCS3.0 CellQuest Pro, FCS Express, WinMDI, ModFit LT FCS2.0 FCS Express FCS Experiment Specimen Tube FCS FCS 2. File > Export > Export Export FCS 3. a. Log 1,024(10-bit) 262,144(18-bit) 18-bit b. FCS2.0 FCS3.0 FCS2.0 Pro 16 FCS2.0 CellQuest 8 FCS2.0 CellQuest CellQuest Pro BD FACSConvert PC Mac FLX-Area 77

78 c. FCS OK 5. Save Export 6. Save 78 BD FACSCanto

79 7. Window Explorer 2.2 FCS FCS2.0 FCS3.0 Instrument configuration FCS File > Import > FCS files 3. 79

80 4. Import 5. Specimen Tubes FCS BD FACSDiVa FCS2.0 Inspector Compensation 80 BD FACSCanto

81 2.3 BD FACSDiVa FCS FCS FCS FCS 1. (+) Browser 2. File > Export > Experiments 3. Export Experiments Delete experiments after export OK 81

82 1. File > Import > Experiments Import 82 BD FACSCanto