3-Color FACSCan

Optical Bench

Before you decide on a fluorochrome for a flow cytometric assay, you need to know:
  • the wavelengths of the lasers in the cytometer
  • the wavelengths of the filters in front of the photomultiplier tubes (PMTs) in the cytometer
  • the absorption and fluorescence wavelengths of fluorochromes to be used

These can be simulated using the BD fluorescence spectrum viewer

Technical Specifications

Performance

Fluorescence sensitivity

< 2,000 molecules of equivalent soluble fluorescein

Fluorescence resolution

< 3% for PI labeled chick erythrocyte nuclei

Forward and side scatter sensitivity

Sensitivity enables the separation of fixed platelets from noise

Forward and side scatter resolution

5,000 events/sec

Excitation optics

Optical platform

Fixed optical assembly

Laser

20mW Coherent 488nm, air cooled, argon ion laser

Emission optics

Optical Coupling

Quartz cuvette is coupled to emission lens by refractive index-matching optical gel for optimum collection efficiency

Three fluorescence detectors

High performance, high dynamic range photomultipliers with filters for FITC, PE and PerCP or PE-Cy7

Signal processing

Workstation resolution

Four decades for peak detection

Data acquisition channels

Five acquisition channels

Dynamic range

12-bit data acquisition
1,024 linear channels displayed in a 4-decade logarithmic display

Pulse processing

Not available

Time

Time available correlated to any parameter

Analogue Compensation

Electronics provides analogue correction of spectral overlap

Data management

Central processing

Intel processor

CellQuest v3.3

Apple Mac G4 computer with OS 9.2.2 operating system

Monitor

19-inch Dell LCD monitor

Data File Structure

Flow Cytometry Standard 2.0

Adapted from http://www.icms.qmul.ac.uk/flowcytometry/instruments/facsan/index.html

How to Run the FACScan

Prepare the flow cytometer (check fluidics - sheath fluid level, bubbles, waste container level);

Open a CellQuest software template to run the flow cytometer;

Acquire your unstained control cells;

Acquire your compensation control beads (or cells);

Acquire your positive control cells (fully stained sample);

Check daily Flow Core QC and ensure your PMT settings are similar to QC settings;

Acquire your sample tubes;

Clean the flow cytometer (RHSSCRAQ);

Save your files to a memory stick;

Close CellQuest;

Remove your gloves, tubes and other belongings and clean up any trash;

Sign up for next appointment;

Analyse your Data on any of the work station computers.

If you need any explanation of any of these procedures, please ask Gary Ward.



A detailed description of how to run a FACScan can be found on the excellent website for the FlowLab at the Instituto Gulbenkian de Ciencia.

When you have finished using the FACScan or Calibur, you must follow the 5 steps of the RJRHSSCRAQ Procedure. Otherwise you could run the sheath tank dry or fill the fluid lines with bubbles or clog the flow cell.

1) Run dilute JetDry for 2 minutes;

2) Run distilled H2O for 2 minutes;

3) Switch to Standby and remove the sheath pressure (push Valve to the back). Install a sample tube with 2 ml (NOT MORE) of distilled water.

4) Check Reservoirs; fill or empty as required. Please clean up spills.

5) Quit CellQuest.