BD FACSCanto II. Tel :

BD FACSCanto II http://www.bd.com/tw/ Tel : 02-27225660

BD FACSCanto II BD FACSCanto 03 BD FACSCanto 13 Surface Markers.. 22 DNA Analysis... 34 FACSDiva 47. 58 2

BD FACSCanto II BD FACSCanto II 488nm 20mw 633nm 17mw 6~8 2 0.1% 120 ul/min 10,000 events/sec BD FACSCanto II 3

1.1 BD FACSCanto II BD FACStation FACSCanto II 4

1.2 Fluidics Sample injection tube (SIT) aspirator arm flow cell SIT SIT flow cell tubing SIT aspirator arm aspirator arm bar SIT tube guide SIT (Loader ) tube eject cylinder (Loader ) tube sensor SIT bal seal access Bal seal BD FACSCanto II 5

SIT SIT SIT SIT BD FACSDiva SIT Acquisition Dashboard SIT Flush Checkbox SIT FACSCalibur 3 ml 1.2.1 Flow Cell 6

1.3 Optics BD FACSCanto II 1.3.1 Excitation Optics 1-3 FACSCanto II BD FACSCanto II nm mw Coherent R Sapphire TM 488 20 FITC, PE, PE-Texas Red, PerCP, PerCP-Cy5.5, PE-Cy7,PI JDS Uniphase TM HeNe 633 17 APC,APC-Cy7 ( 4-2-2 ) BD FACSCanto II 7

1.3.2 Collection Optics ( 1-3) 4-2 FACSCanto 1-4 90 ( 4-2 ) Longpass DM PMT PMT PMT bandpass Blue A 750~810 nmblue B 670~730nmBlue D 564~606nmBlue E 515~545nmBlue F 488nm 8

photodiode ( 1-2) ( 4-2 ) PMT Position LP Filter nm BP or LP Filters Fluorochromes nm 488 Laser Blue A: 735 780/60 PE-Cy7 Blue B: 655 670LP PerCP, PerCP-Cy5.5 Blue C: 610 Blank filter N.A Blue D: 556 585/42 PE Blue E: 502 530/30 FITC Blue F: Blank filter 488/10 SSC 633 Laser Red A: 735 780/60 APC-Cy7 Red B: 685 Blank filter N.A Red C: Blank filter 660/20 APC BD FACSCanto II 9

1.4 Electronics PMT BD FACSCanto II BD FACSCanto User s Guide Signal detection 1.4.1 Power Panel Port or Button System Power FACSCanto II Power Out FACSCanto II Communication cable System AC power plug System circuit breaker 10

1.4.2 Acquisition Indicator Lights BD FACSCanto II 11

1.5 1.5.1 20 FACSFlow 10 5 FACS Shutdown 5 FACSClean BD FACSCanto BD FACSDiva 12

1.5.2 FACSCanto II Off Circuit Breaker On Powering Off Condensation Trap 60~70psi 50psi BD (0800-737-842) BD FACSCanto II 13

1.5.3 1.6 BD FACSCanto II BD FACSCanto II FACSCanto II BD FACSCanto II Clinical or FACSDiva BD PC Pentium R IV 1GB RAM 40GB Windows XP Professional (US-English) 17 BD FACSCanto II Clinical Software version 2.1 or above Instrument QC, Lymphocyte subset TBNK, HLA-B27, Stem Cell Enumeration BD FACSDiva version 5.0 or above BD FACSDiva Data Manager 14

Cytometer > Fluidics Startup OK BD FACSCanto II 2.1 1. BDIS ( )Enter 2. 3. 4. BD FACSDiva 5. Instrument 6. Instrument Instrument Connected Instrument FACSFlow Instrument Disconnected FACSDiva 7. : Shutdown solution Sheath buffer System Prime -- Bubble filter purge Degas flow cell 5~10 BD FACSCanto II 15

Instrument > Automatic Cleaning FACSDiva FACSDiva Automatic Clean Automatic Clean 8. OK : For Diva 6.0 9. Instrument Remaining warmup time 7:27 The system is ready 10. 16

2.2 (Controls in Instrument Frame) Fluid Level Indicators BD FACSDiva FACSFlow 17% 34% 51% 68% shutdown cleaning 20% FACSFlow FACSFlow ( ) ( ) FACSFlow <17% >83% FACSFlow 0% >99% Laser Tab Window Extension FSC Area Scaling BD FACSCanto II 17

Status Tab 2.3 Acquisition Dashboard Next Tube Acquire Data Record Data list mode data Restart Acquire Record SIT Flush SIT 18

Flow Rate Low 10 µl /min Medium 60 µl /min High: 120 µl /min 2.4 1. Fluidics Startup 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. Basic controls Acquire Data 3. High Flow Rate Acquire 5 5 Acquire 4. FACSClean 3 ml dh2o BD FACSCanto II 19

5. Acquisition controls Acquire High Flow Rate Acquire 5 6. 5 Acquire Fluidics Shutdown 7. Cytometer > Fluidics Shutdown OK OK Automatic Cleaning FACSDiva Fluidics Shutdown 1 ml SIT 8. dh2o 9. 1/10 10. Condensation Trap 20

2.6 FACSCanto II Clean Flow Cell.. Instrument/Clean Modes/Clean Flow Cell.. 3 ml FACSClean FACSClean fluidics startup fluidics shutdown BD FACSCanto II 21

De-Gas or Bubble Filter Purge 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 22

2.7 FACSCanto II BD FACS 7-color setup beads Cat. No. 335775 Rainbow fluorescent particles Cat. No. 556288 Nile Red fluorescent particle Cat. No. 347240 BD FACSFlow sheath fluid Cat. No. 342003 BD FACS cleaning solution Cat. No. 340345 BD FACS shutdown solution* Cat. No. 334224 House hold bleach for waste 5mL polystyrene test tubes (BD Falcon ) uncapped, 125 per bag BD Cat. No. 352052 capped, 125 per bag BD Cat. No. 352054 capped, 25 per bag BD Cat. No. 352058 with cell-strainer cap BD Cat. No. 352235 *BD FACS shutdown solution BD FACSCanto II 23

Surface Markers BD FACSDivia Experiment Global Sheet 24

Surface Markers Instrument Setting ( ) 3.1 BD FACSDivia 1 2 BD FACSDivia 3 View View>>Reset positions 3.2 4 Browser ( ) >> >> >> (Folder>>Experiment>>Specimen>>Tube BD FACSCanto II 25

5 Folder Folder 6 Experiment Folder Experiment Experiment>>New Experiment Experiment templates Blank Experiment with Sample Tube 7 FACSDiva Blank Experiment with Pop-up Menu 2-color exp Enter 26

8 Blank Experiment with Sample Tube Specimen_001 Specimen Specimen Specimen Tube_001 Specimen_001 Tube 3.3 9 2-color exp Instr Settings ( ) Inspector Parameters Parameters PerCP-Cy5-5 PE-Cy7 APC APC-Cy7 FITC PE Parameters Add or Delete FITC EGFP Alexa 488 PerCP-Cy5-5 PerCP PI 3.4 Global Sheet BD FACSCanto II 27

10 Global Sheet1 Dot Plot 1 FSC-A vs SSC-A 2 FITC-A vs PE-A 3.5 Optimize Settings FSC SSC Surface Markers CD3 FITC 3 CD4 PE FSC SSC 11 Instument Tube_001 12 Basic Controls Acquire Record 28

FSC SSC Voltage Instrument Parameters FSC SSC FSC SSC Acquire 13 Basic Controls Acquire Instrument Threshold FSC 14 Global Sheet1 FSC-A vs SSC-A P1 P1 BD FACSCanto II 29

15 Shift Show Population P1 P1 Instument Voltage 16 Tube_001Basic Controls Acquire Record Instrument Parameter Acquisition Controls Acquire FITC-A vs PE-A quadrants 30

17 FITC Tube_001 Basic Controls Acquire PE-%FITC unstained(q3) FITC alone(q4) PE-A Mean 18 PE Tube_001 Basic Controls Acquire FITC-%PE unstained(q3) PE alone(q1) FITC-A Mean BD FACSCanto II 31

19 Experiment Instr Settings Inpector Print Typical Instrument Setting for 2-color experiment on FACSCanto II 3.6 20 Specimen_001_Tube_001 Specimen_001 Inspector Sample_0831 Enter 32

21 Tube_001 Inspector Name CD3_CD4_001 Enter Diva 22 Labels Lable ( ) FITC Label CD3 23 Lable ( ) Events to record Stopping gate Storage gete Global sheet1 FSC/SSC P1 populations marker quadrants 24 CD3 FITC/CD4 PE Sample_001 CD3_CD4_001 Acquisition Controls Acquire P1 Acquisition Controls Record BD FACSCanto II 33

25 10,000 25 Acquisition Controls Next Tube Tube CD3_CD4_002 CD3_CD4_002 CD3 FITC/CD4 PE Acquisition Controls Acquire P1 Record 26 34

DNA Analysis DNA BD FACSDivia DNA Analysis Global Sheet ModFit LT BD FACSCanto II 35

DNA Instrument Setting ( ) 4.1 BD FACSDivia 1 2 BD FACSDivia 3.1.1 For Diva 4.X.X or 5.X.X : Instrument>Instrument Configuration DNA PI DAPI Duplicate DNA_001 OK 3.1.2 Parameter Add PI Blue (Laser) D(Detector) OK 3.1.3 Set 36

3.2.1 For Diva 6.0 or above : Cytometer>View Configurations DNA PI DAPI Configuration Duplicate DNA_001 3.2.2 Edi 3.2.3 Ctrl Parameters PE PI Blue D 3.2.4 Set Configuration OK 4.2 DNA Analysis 7 Folder Folder 8 Experiment Folder Experiment Experiment>>New Experiment Experiment templates Blank Experiment with Sample Tube BD FACSCanto II 37

9 FACSDiva Blank Experiment with Inspector DNA_001 Enter 10 DNA_001 Specimen_001 Specimen Specimen Specimen Tube_001 Specimen_001 Tube 4.3 11 DNA_001 Instr Settings ( ) Inspector Parameters Parameters PerCP-Cy5-5 PE-Cy7 APC APC-Cy7 FITC PE 12 Parameters Add or Delete PE P 38

13 PI LIN PI-W 4.4 Global Sheet 14 Global Sheet1 Dot Plot 1 FSC-A vs SSC-A 2 PI-W vs PI-A Histogram 3 PI-A histogram BD FACSCanto II 39

4.5 Optimize Settings PI=5,000 DNA FSC SSC optional DNA Unstimulated PI Stimulated Positive PI PI 15 Tube_001 Instrument Threshold PI PI 5,000 16 DNA Low 40

17 Basic Controls Acquire Record PI Voltage G0 PI-A 50 X1000 18 Global Sheet1 PI-W vs PI-A Single Cells P1 P1 FSC SSC 19 Instrument Parameters FSC SSC FSC SSC FSC SSC Voltage Jurkat Acquire BD FACSCanto II 41

20 PI-A Show Population P1 PI-A P1Jurkat 21 Basic Controls Acquire Instrument Threshold PI 22 DNA Experiment Instr Settings Inpector Print 42

4.6 25 Specimen_001_Tube_001 Specimen_001 Inspector Sample_0831 Ente 26 Tube_001 Inspector Name PI_001 Enter Diva 27 Labels Experiment>Experiment Layout Experiment Layout Lable ( ) 28 Experiment Layout Acquisition ( ) PI_001 Events to Record 20,000 OK BD FACSCanto II 43

29 Acquisition Controls Low Stopping gate Storage gete 30 Global sheet1 PI-W/PI-A P1 P1 31 PI Sample_001 PI_001 Acquisition Controls Acquire P1 Acquisition Controls Record 20,000 32 Acquisition Controls Next Tube Tube PI_002 PI_002 Acquisition Controls Acquire P1 Record 33 44

4.7 34 DNA ModFit LT Cell Cycle DNA_001 File>>Export>>FCS files 35 FCS2.0 BD FACSCanto II 45

36 Cell Cycle G0/1-S-G2/M ModFit LT 46

FACSDiva FACSDiva BD FACSDvia Global sheet BDISEnter BD FACSDiva 5.1 1 Experiment>>New Experiment Experiment templates Blank Experiment BD FACSCanto II 47

2 File>>Import>>FCS Files D:/BD EXPORT/EXPERIMENT/6 Color cocktails 4 5.2 Global Worksheet 3 New Global Worksheet Global Sheet2 Analysis 48

4 Analysis 1 FSC-A vs SSC-A 2 FITC-A vs PE-A 5 tube_001 Analysis FSC-A X FSC-H BD FACSCanto II 49

6 snap-to-gate FSC-A SSC-A Diva 7 FITC-A PE-A Show Populations P1 8 FITC-A PE-A P1 50

9 Create statistics View 10 Edit Statistics View 11 P1 Q1~4 BD FACSCanto II 51

12 Statistics 5.3 13 Analysis File >> Print 5.4 14 CSV File >> Export >> statistics 52

15 test1 Save 5.5 16 Shift 17 File >> Export >> Worksheet Elements 18 Expor BD FACSCanto II 53

5.6 19 P1 20 6 Color LnL Inspector Analysis 21 6 Color LnL Batch Analysis Print Statistics START 54

22 test 1 Save BD FACSCanto II 55

5.7 Log Out 23 - FACSDiva 24 File >> Quit File >> Log Out Quit 5.8 Excel 25 Excel >> Microsoft Excel 26 D:\BD Export\Statistic CSV 56

27 30 BD FACSCanto II 57

(PMTs 4-2 ) (nm) (nm) Fluorescein, FITC 490 520 (Blue E) Phycoerythrin-R, PE 495 578 (Blue D) Peridinin-chlorophyll, 490 677 (Blue B) PerCP PerCP-Cy5.5 490 (Blue B) Cy-Chrome 495 670 (Blue B) PE-Texas Red 495 620 (B or D) PE-Cy5 495 670 (Blue B) PE-Cy7 490 781 (Blue A) Allophycocyanine 650 660 (Red C) APC-Cy7 650 781 (Red A) Ethidium Bromide 510 (+ds DNA) 595 (Blue D) Propidium Iodide 536 (+ds DNA) 623 (B or D) Acridine Orange 480 (+ DNA) 440-70 (+ RNA) 520 (Blue E) 650 (Blue B) Thiazole Orange 509 (+ RNA) 533 (Blue E) Viability Propidium Iodide 536 623(B or D) YOPRO-1 480 515 (Blue E) Acridine Orange 480 520 (Blue E) 7-AAD 488 670 (Blue B) DiO-C6 (3) 485 510 (Blue E) Mitochondrial Rhodamine 123 485 546 (Blue E) ph BCECF-AM 488 Ratio 520/620 SNARF1-AM 514 Ratio 587/640 Fluo4-AM 488 528 (Blue E) Calcium Green-1 488 530 (Blue E) Fura Red 488 660 (Blue B) H2O2 sensitive Dihydrorhodamine 123 505 534 (Blue E) DCFH-DA 505 535 (Blue E) O2- radical sensitive Hydroethidine 505 600 (Blue D) Esterase sensitive Fluorescein -DA 495 525 (Blue E) 58

Optical Configurations nm mw Coherent R Sapphire TM 488 20 FITC, PE, PE-Texas Red, PerCP, PerCP-Cy5.5, PE-Cy7, PI JDS Uniphase TM HeNe 633 17 APC, APC-Cy7 Point Source iflex2000 405 30 Pacific Blue, AmCyan ( 4-2-2 ) PMT Position LP Filter nm BP or LP Filters Fluorochromes nm 488 Laser Blue A: 735 780/60 PE-Cy7 Blue B: 655 670LP PerCP, PerCP-Cy5.5 Blue C: 610 Blank filter N.A Blue D: 556 585/42 PE Blue E: 502 530/30 FITC Blue F: Blank filter 488/10 SSC 633 Laser Red A: 735 780/60 APC-Cy7 Red B: 685 Blank filter N.A Red C: Blank filter 660/20 APC 405 Laser Viiolet A: 502 510/50 AmCyan Violet B: Blank filter 450/50 Pacific blue BD FACSCanto II 59

( 5-3 ) PMT Position LP Filter nm BP or LP Filters Fluorochromes nm 488 Laser Blue A: 735 780/60 PE-Cy7 Blue B: 655 670LP PerCP, PerCP-Cy5.5 Blue C: 610 616/23 PE-Texas Red Blue D: 556 585/42 PE Blue E: 502 530/30 FITC Blue F: Blank filter 488/10 SSC 633 Laser Red A: 735 780/60 APC-Cy7 Red B: 685 712/21 Alexa Fluor-700 Red C: Blank filter 660/20 APC 60