4-Color FACSCalibur

 

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


Specification

Comments

Performance

Fluorescence sensitivity

750 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

Optimized scatter performance for resolving lymphocytes, monocytes, and
granulocytes.

Excitation optics

Optical platform

Fixed optical assembly

Lasers

15mW Coherent 488nm, air cooled, argon ion laser
635nm red laser

Emission optics

Optical Coupling

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

Four fluorescence detectors

High performance, high dynamic range photomultipliers (PMTs) with bandpass filters: 530 nm (FITC), 585 nm (PE/PI), and >670 nm (PerCP, PE-Cy5, PE-Cy7) and 661 nm (APC)

Sample concentration

Single-cell suspension of 105 to 2 x 107cells/ml recommended range.

Sample flow rates

Three selectable flow rates of 60 μl/min, 35 μl/min, and 12 μl/min;
Particle velocity in flow cell: approximately 6 meters/second.

Signal processing

Dynamic range

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

Pulse processing

Width and area measurements for discriminating doublets; available for all
fluorescence parameters.

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 (FCS) 2.0/3.0

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

More information: FacsCaliburTechSpec.pdf

How to Run the FACScan or the FACSCalibur

IMPORTANT: TURN CYTOMETER ON BEFORE COMPUTER.

GIVE CYTOMETER 30 MINUTES TO WARM-UP BEFORE USE.

 

Before Acquisition

1

Switch On the FACScan with the black switch on the left front panel;

2

Switch on the computer immediately afterwards;

3

Fill the PBS reservoir. Please clean up all spills;

4

Start CellQuest by clicking on the icon for a stored CellQuest template or by clicking on the CellQuest software application icon on the desktop;

5

Verify that the PBS filter is free of airbubbles. If not, vent the filter by slightly tapping while opening and closing the bleed valve. In case of air bubbles you need to run the machine on PBS; (link ‘air bubbles’ to FACSCAN Drain/Fill below);

6

Pressurize the PBS reservoir (pull Valve down);

7

Put the PBS Sample Tube with the support lever to the right and aspirate 2 ml of PBS. Move the lever to secure the tube;

8

Put the machine speed on high and select RUN;

9

Start CellQuest by clicking on the icon for a stored CellQuest template or by clicking on the CellQuest software application icon on the desktop;

10

Acquire -> Connect to Cytometer;

11

Acquire -> Counters;

12

Acquire -> Parameter Descriptions; use ≥folder≤ to set location and ≥file≤ to set name for data storage;

13

Acquire -> Acquisition and Storage to set number of cells to be saved;

14

Cytometer -> Instrument Settings, to get previously-stored electronic settings;

15

Click ≥Open≤ to find an appropriate stored settings file; then ≥Open≤ it;

16

Click ≥Set≤ to apply these settings to the cytometer. Watch for the globe, then click ≥Done.≤;

17

Press apple1 to show/alter detector settings;

18

Press apple2 to show/alter the threshold;

19

Press apple3 to show/alter the compensation;

20

Set up your plots;

21

Open the Acquistion Control window (Cytometer Status) and check if status is STANDBY, sheath fluid is OK, and sample voltage is approx. 10.23. If not, make sure PBS tank is well tightened, or check troubleshooting instructions;

22

When status switches to STANDBY, run PBS for about 1min in HIGH to check if machine is dirty. Though events may appear, it may not be dirt but air bubbles being released. Still, these are events that may or may not interfere with acquisition.
Evaluate noisy events based on the location where they fall in your plots and on the rate at which you will be acquiring your sample;

23

If too many events appear, it is probably due to the machine being dirty. Follow Cleaning Procedures in the After Acquisition section, and then repeat the previous step;

24

You are ready to start Acquisition!

 

After Acquisition RHSSCRAQ

1

When done, first run 10% bleach for 2 minutes if you have used a DNA dye (eg. PI, 7-AAD, ToPro3);

2

Everyone must run diluted JetDry for 2 minutes, followed by distilled water for 2 minutes;

3

Leave 1 ml of water on the probe;

4

Switch to standby and remove pressure (pull valve up);

5

REFILL PBS tank and replace PBS tube with H2O tube;

6

Quit your acquisition files and REMOVE all your data;

7

If you are the last user of the day, continue with shut-down instructions.

 

Shut-down Instructions

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 Cell Quest.

 

Drain and Fill Procedure to Remove Bubbles

1

Check to make sure that waste tank is not full. If it is, it needs to be emptied into the sink and the sheath tank needs to be filled with FACS buffer from the box;

2

Look for bubbles in the Pall Filter (spherical plastic sheath filter inside lower case). This must be purged of air before a successful "drain and fill" may be accomplished. This filter is rubber mounted and may be tapped lightly to bring air bubbles to the top. The bubbles may then be drained into a beaker after the white plastic cap is removed from the rubber tubing. WARNING! Contents under pressure! Have beaker in place before removing cap;

3

Remove tube from the sample probe;

4

Place the fluidics knob (black knob lower right hand corner of cytometer) in the "BACK FLUSH" position for 20 seconds;

5

Place the fluidics knob in the "DRAIN" position. Watch the waste line (1/4 inch Tygon tubing with orange connector inside lower case) to see when fluid has drained;

6

After fluid has drained place fluidics knob in "fill" position. Watch the quartz cuvette (top right hand corner inside upper case) to make sure the meniscus is level and there are no air bubbles or obstructions in the fluid as it rises;

7

If an uneven fluid level is present or if there is an air bubble or obstruction in the quartz cuvette repeat from 4 above;

8

After quartz cuvette has evenly filled, watch waste line to see that a continuous flow of sheath is moving. An occasional air bubble here is OK;

9

Place fluidics knob in "run" position to continue sample acquisition.

 

REMOVE your data immediately by transferring the files to another computer over the network or to Zip disks (the computer does not support memory sticks). Remember that the FACScan hard drive is NOT for data storage!

Switch OFF the FACSCalibur at the right side.

Switch OFF the computer by choosing "Shutdown" form the Menu "Special". Switch off the MO Drive on the RED Switch of the multiconnector

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

Analyse your Data on any of the work station computers.

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

The best way to store your own data is to transfer it to your directory. Follow the following steps:

  1. Open the Chooser (click the apple on the upper-left corner).
  2. Select AppleShare.
  3. Click on "Server IP Address..."
  4. Enter the name of the remote server you want to connect to.
  5. Click Connect.
  6. Enter your username and password and click Connect again.
  7. Select the folder you want to connect to (DO NOT CLICK THE CHECKBOXES!!)
  8. Your folder will appear on the Desktop as a disk with your folder's name.
  9. Transfer your files by dragging them into the remote folder.
  10. Delete the original data on the local computer.
  11. To disconnect from the remote server drag the remote disk into the trash.

You can also try to save your data directly onto a ZIP disk. If you wish to burn a CDR you can do this directly on the FACSCan. See Topic on How to burn CDs in the Computer Section. Always burn in duplicates and always use reliable brands for the CDRs (Philips, Sony, Maxell, Verbatim etc).

For more details go to the excellent site Acquiring with Cell Quest interface.