CyAn : 11 Parameter Desktop
Flow Cytometer
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
Cyan ADP
Cyan ADP
Spatially separated laser Lines – reduced crosstalk
Cyan ADP
Which fluorochromes can we detect?
2-3 lasers: 488nm, 635nm and 405nm
635nm 405nm 488nm
Cyan ADP
Easy to Change
Filters & Dichroics
Cyan ADP - Control
Cyan ADP controlled by the Summit
Software
1. Fluidics
2. Lasers
3. PMTs
Cyan ADP - Control
•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
1.Fluidics
Cyan ADP - Control
Instrument control panel
•Turn Lasers On and Off
•Control Laser Shutters
Summit Software
Cyan ADP - Control
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
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
Cyan ADP - Software
•
Boolean gates
•
Overlays
•
Subtraction
•
Normalisation
•
Multiple Sample
Analysis
•
Zoom
Cyan ADP - Software
•Drag and Drop
•9 x 9 Compensation Matrix •Compensation pre- and
post-acquisition
•Help function
•Smoothing/Contours
•Colours/Fonts •Exportable
Cyan ADP - Applications
•
Multicolour immunophenotyping
•
Rare event detection
•
High speed immunophenotyping
•
Small Particle analysis
•
Signal Transduction
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.
Cyan ADP - Mutlicolour
immunophenotyping
0 64 128 192 256 FSC 0 64 128 192 256 SSC Granulocytes Monocytes Lymphocytes Debris Eosinophils R6 100 101 102 103 104 CD 3 PE-TR Comp 0 64 128 192 256 SSC T - Lymphocytes SSC vs. FSC SSC vs. CD3 PE-TR Leukocyte PopulationsCyan ADP – Multicolour
immunophenotyping
Leukocyte Populations R2 R3 R4 R9 R11 100 101 102 103 104 CD 45 FITC Comp 100 101 102 103 104 CD 1 4 P E -Cy 5 Co m p Monocytes Lymphocytes Eosinophils Neutrophils BasophilsCD14 PE-Cy5 vs. CD45 FITC SSC vs. CD45 FITC
R1 100 101 102 103 104 CD 45 FITC Comp 0 64 128 192 256 SS C Lymphocytes
Cyan ADP – Multicolour
immunophenotyping
B and T cell populations
R12 R13 R14 R15 100 101 102 103 104 CD 3 PE-TR Comp 100 101 102 103 104 CD 19 A P C Com p Gated on Lymphocytes B Cells T Cells CD19 Positive Lymphocytes APC positive (FL8) Excitation: 635 nm Detection: 665/20 BP In this donor, 13.0% of the lymphocytes are B cells.
CD19 APC vs. CD3 PE-TR
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.
R11 R12 R13 R14 100 101 102 103 104 CD 56 PE Comp 100 101 102 103 104 C D 19 A P C Com p CD19 APC vs. CD56 PE Gated on Lymphocytes NK cells CD56 Positive Lymphocytes PE positive (FL2) Excitation: 488 nm Detection: 575/25 LP In this donor, 0.36% of the lymphocytes are NK.
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
Cyan ADP – High speed
analysis
High Speed Immunophenotyping
1 x 106 cells/ml 500,000 events 8 min 18 sec 5 x 106 cells/ml 500,000 events 1 min 38 sec 50 x 106 cells/ml 500,000 events 10 seconds
Cyan ADP – Small particle
analysis
Small particle
analysis
Advantage of distinguishing
different species of
bacteria by light scatter
One customer is even looking
at levels of bacteria &
yeast in wine
100 101 102 103 104 [2165] 106.3% (FSC) 100 101 102 103 104 [246 9] 125. 0% ( S S C ) Bacteria spores, 0.5 µm Bacteria, 1-2 µm Bacteria spores, 0.8 µm Fungus, 5 µmCyan ADP – Signal
transduction CFP/YFP FRET
Transfected cells CFP only
Transfected cells YFP only
Transfected cells FRET
FSC v SSC gate
Cyan ADP - DNA cell viability
and cycle analysis
DNA Dyes
488 Excited