Institute of Immunology & Infection Research

Flow Cytometry Core Facility

FACSCalibur(BD) Bench-top Analyser

- dual laser 488nm argon and 633nm red diode
- 6 parameter
- routine cytometer for 4 colour analysis


Performance  (Top)
Fluorescence Sensitivity Estimated detection limit is 750 molecules of equivalent soluble Fluorescein
Fluorescence Resolution Coefficient of variation in FL2-Area of < 3%, full peak for propidium iodide–stained chicken erythrocyte nuclei
Forward and Side
Scatter Sensitivity
Sensitivity enables the separation of fixed platelets from noise
Forward and Side
Scatter Resolution
Scatter performance is optimized for resolving lymphocytes, monocytes, and granulocytes
Acquistion rate 4,000 events/second(max)

Excitation Optics  (Top)
Optical Platform Fixed optical assembly
Lasers 15 mW 488 nm, air-cooled argon-ion laser
12mW second red diode laser: nominally 635 nm

Emission Optics  (Top)
Optical Coupling Quartz cuvette is coupled to emission lens by refractive index matching optical gel for optimum collection efficiency
Background Rejection Obscuration blade and slit minimize unwanted laser radiation at the detector
Forward Scatter Detector and Filter High-performance solid-state silicon detector; 488 nm bandpass filter for clear signal detection and red diode laser signal rejection
Side Scatter Detector Photomultiplier using Brewster-angle beam splitter in the emission optical train
Fluorescence Detectors and Filters High performance, high dynamic range photomultipliers with bandpass filters
488nm
Fluorescence Detectors and Filters

530/30nm      FITC / Alexa-488 / GFP / CFSE
585/42nm      PE / DsRED / PI
 > 670nm       PE-Cy5 / PerCP / PI / 7AAD
                      PerCP-Cy5.5 / PE-Cy5.5 / PE-Cy7
633nm
Fluorescence Detectors and Filters

661/16nm       APC / Alexa-633 /Alexa-647
 


Fluidics  (Top)
General Operation Front key panel control provides three modes: RUN, STNDBY, and PRIME; automatic standby mode conserves sheath fluid by stopping sheath flow when no sample tube is installed
Fluid Reservoirs Easily accessible 4L capacity sheath and waste containers are housed in a convenient pull-out drawer; level detectors automatically indicate low levels of sheath or high levels of waste
Sample Flow Rates Three selectable flow rates of 60 µL/min, 35 µL/min, and 12 µL/min. Pressure difference between sheath and sample is regulated and monitored; particle velocity in flow cell is approximately 6 meters/sec
Quartz Cuvette Internal cross-section is rectangular 430 x 180-µm; external surfaces are anti-reflection coated for maximum transmission of laser light
Sample Concentration
Single-cell suspension of 105 to 2 x 107 particles per mL recommended range

Signal Processing  (Top)
Workstation Resolution 1024 channels on all parameters
Dynamic Range Logarithmic amplifiers for SSC, FL1, FL2, FL3, and FL4 provide four log decade range
Fluorescence Compensation Networks Fluorescence spectral overlap can be compensated between FL1 and FL2, between FL2 and FL3 channels, and between FL3 and FL4 
Pulse Processing Width and Area measurements for discriminating doublets; available for all fluorescence parameters
Time Time available correlated to any parameter for kinetic experiments or other applications.

Data Management System   (Top)
Workstation Macintosh G5
Central Processing Unit (CPU) Power PC Reduced Instruction Set Computer (RISC) CPU
Memory 500MB RAM
Level 2 Cache 256 kilobytes
Data Storage 1.2 gigabyte hard disk
Networking On-board Ethernet, built-in AppleTalk Networking, and File Sharing
CD ROM 4x Speed CD 600i
Monitor 17-inch MultiSync Monitor
Data File Structure Flow Cytometry Standard (FCS) 2.0 ASCII results file for data export