FACSCalibur Training General Information. The FACSCalibur is a useful analysis tool. The instrument has 2 lasers- 488 (primary) and 633 (secondary) that can detect FSC, SSC, FL-1 (FITC) FL-2 (PE) FL-3 (CYC, Tri-Color) and FL-4 (APC).
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CD3 is a T-cell marker
CD19 is a B-cell marker
Laser Point of Interrogation
488nm and 633nm lasers
P1 FSC Forward Scatter
P2 SSC Side Scatter
Both are 488 excitable
The 2 most important parameters
Parameter Detector Excitation Emission
P3 FL-1 FITC 488 518
P4 FL-2 PE 488 575
P5 FL-3 CYC 488 667
P7 FL-4 APC 633 660
Excited by a discreet wavelength from the excitation source (laser), the fluorochrome will not emit a discreet wavelength of light, but rather a spectrum, often a very broad spectrum.
FITC Overlapping into the PE Channel
FL-2 (-) % FL-1 ~ 20-30%
PE overlapping into FITC, but not by much
FL-1 (-) % FL-2 ~ 0.1-1.0%
Starting Up CellQuest Pro and Connecting to the Cytometer
Once the instrument is warmed up and ready for use, a QC procedure called “Time Delay Calibration” must be performed. This will allow the lasers to fire in their appropriate time in space. This must be done every time a user turns on the machine.
Instrument Quality Control- Time Delay Calibration
1. Go to File> Choose “Open Document” and then look in Data 1> Setup Folder> and choose “Time Delay Calibration”.
Now the machine is ready for use. The QC has been performed and you are now ready to use the instrument for your analysis needs. Located below is a brief description of how to set up a protocol and how to label your folders and file names.
Creating a New Protocol
4. You can also highlight a plot and go to “Stats” and then choose “Histogram” or “Quadrant” stats based on the plot type. The software allows you to edit the stat boxes to display any number of items that are pertinent to you. Remember- the simplest experiment is the best experiment.
5. Once you have everything in place and to your liking, go to “File” and choose “Save Document As” and then navigate to your investigators folder and give your protocol a name that you can remember and one that fits the protocols needs e.g. “FITC-PE”, “Cell Cycle”, or “Dendritic Cells with DsRed” or whatever best describes your experiment so you can remember it.
Naming of Folders and files is very important. This will allow you to go to your Investigators folder and find your data easily for future analysis.
Naming of Folders and Individual Files
3 letter month abbreviation
Today's date and year
Folder name= C1WCAUG1108
File name= C1WC081108
Everything is now in place for you to acquire your data. Take the time to make sure that the folders and files are labeled correctly and that your protocol is exactly the way that you want it. Making sure these things are correct will save you time and frustration later.
In the careful planning of your experiment, you have determined that controls are needed. Controls will allow you to set up the instrument with regards to baseline fluorescence and compensation.
Negative and Single Positive Controls- Compensation
Single + Control for each fluorochrome used
Ex. If you have a 3 color experiment you need 4 total controls.
5. As a guideline- the FL-2 - % FL-1 is roughly 20-30%. The FL-1 – % FL-2 is roughly 0.1-1.0%. However, these are just guidelines and vary based on assay being performed, staining technique, antibody concentration, etc.
****You need to have single positive controls for all the colors you are using including a negative. The more controls that you have the better your experiment will turn out.
6. Once you have run all your controls you can proceed and acquire the rest of your data.
7. After you are done with your run place a tube of water on the instrument (sample injection port) and place the machine in standby. You are free to go on about your day. Nothing else is required of you.
To shut the machine down: