CyAn : 11 Parameter Desktop Flow Cytometer

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1 CyAn : 11 Parameter Desktop Flow Cytometer

2 Cyan ADP 3 excitation lines 488nm, 635nm, and UV or violet 11 simultaneous parameters FSC, SSC, and 7-9 colors with simultaneous width, peak, area, and log on each High Throughput >50,000 events/sec analysis rate Desktop footprint

3 Cyan ADP Optical layout

4 Spatially separated laser Lines reduced crosstalk Cyan ADP

5 Cyan ADP Which fluorochromes can we detect? 488nm 2-3 lasers: 488nm, 635nm and 405nm 635nm 405nm

6 Cyan ADP Easy to Change Filters & Dichroics

7 Cyan ADP - Control Cyan ADP controlled by the Summit Software 1. Fluidics 2. Lasers 3. PMTs 4. Trigger and Threshold

8 Cyan ADP - Control 1.Fluidics Sheath management system (SMS) 20-24hours sample run time Event Rate- Fully modifiable Backflush Debubble Both software control panel and LEDs on the SMS provide fluid level status information to the user Automated fluidics startup/shutdown and clean/rinse

9 Cyan ADP - Control Summit Software 2. Lasers Instrument control panel Turn Lasers On and Off Control Laser Shutters

10 Cyan ADP - Control 3, 4. PMTs, trigger and threshold CyAn Parameter Panel Edit the Parameter Names Updated in real-time Change the Voltage on the PMT s Change the Lin, Log, Peak or Integral Setting Change the Gain on Linear Settings Change the Threshold

11 Cyan ADP - Software Summit Software Windows platform Powerful data analysis both during and after acquisition Full Control of All Instrument Parameters Ability to Save an Application Protocol Layout Compensation Settings Parameter Names Voltages, Gains, Lin/Log Free Summit Offline

12 Cyan ADP - Software Boolean gates Overlays Subtraction Normalisation Multiple Sample Analysis Zoom Gate limits and Filters

13 Cyan ADP - Software Drag and Drop 9 x 9 Compensation Matrix Compensation pre- and postacquisition Help function Smoothing/Contours Colours/Fonts Exportable Read/Write all FCS formats

14 Cyan ADP - Applications Multicolour immunophenotyping Rare event detection High speed immunophenotyping Small Particle analysis Signal Transduction DNA/cell cycle analysis

15 Cyan ADP Multicolour immunophenotyping 7-Color Staining Panel Monoclonal Antibodies: CD45 FITC Pan Leukocytes CD56 PE NK marker CD3 PE-TR T cells CD14 PE-Cy5 Monocytes CD4 PE-Cy7 T helper CD19 APC B cells CD8 APC-Cy7 T Suppressor/Cytotoxic Preparation: The cell preparation was carried out on human blood, using the Dako Uti-Lyse kit.

16 Cyan ADP - Mutlicolour immunophenotyping 256 SSC vs. FSC SSC vs. CD3 PE-TR 256 Eosinophils SSC 128 Granulocytes Monocytes Lymphocytes SSC 128 T - Lymphocytes R FSC Debris Leukocyte Populations CD 3 PE-TR Comp

17 Cyan ADP Multicolour immunophenotyping CD14 PE-Cy5 vs. CD45 FITC 10 4 SSC vs. CD45 FITC 256 Monocytes R CD14 PE-Cy5 Comp Neutrophils 10 2 R9 R3 R11 Eosinophils Lymphocytes SSC 128 Lymphocytes R4 R CD 45 FITC Comp CD 45 FITC Comp Basophils Leukocyte Populations

18 Cyan ADP Multicolour immunophenotyping CD19 Positive Lymphocytes APC positive (FL8) Excitation: 635 nm B Cells Detection: 665/20 BP In this donor, 13.0% of the lymphocytes are B cells. B and T cell populations CD 19 APC Comp CD19 APC vs. CD3 PE-TR R12 R13 Gated on Lymphocytes CD 19 APC Comp Natural Killer cells CD19 APC vs. CD56 PE 10 4 R11 R Gated on Lymphocytes NK cells R14 R15 R13 R CD 3 PE-TR Comp T Cells CD 56 PE Comp CD3 Positive Lymphocytes PE-TR positive (FL3) Excitation: 488 nm Detection: 619/20 BP In this donor, 74.1% of the lymphocytes are T cells. CD56 Positive Lymphocytes PE positive (FL2) Excitation: 488 nm Detection: 575/25 LP In this donor, 0.36% of the lymphocytes are NK.

19 Cyan ADP Rare event detection Rare event detection High Speed detection of Antigen (EBV) specific tetramers from whole blood (less than 0.1% total blood cells), with very low background CONTROL EBV+

20 Cyan ADP High speed analysis High Speed Immunophenotyping 1 x 10 6 cells/ml 500,000 events 8 min 18 sec 5 x 10 6 cells/ml 500,000 events 1 min 38 sec 50 x 10 6 cells/ml 500,000 events 10 seconds

21 Cyan ADP Small particle analysis Small particle analysis Bacteria spores, 0.8 µm Fungus, 5 µm Advantage of distinguishing different species of bacteria by light scatter One customer is even looking at levels of bacteria & yeast in wine [2469] 125.0% (SSC) Bacteria spores, 0.5 µm Bacteria, 1-2 µm [2165] 106.3% (FSC)

22 Cyan ADP Signal transduction CFP/YFP FRET Transfected cells CFP only Transfected cells YFP only Transfected cells FRET FSC v SSC gate

23 Cyan ADP - DNA cell viability and cycle analysis DNA Dyes 488 Excited Propidium Iodide (PI) 7-AAD UV Excited DAPI Hoechst 635 Excited DRAQ5 CyAn provides great flexibility as to which laser is used for your DNA measurement

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