Standard fluorescent beads are used to check performance and alignment of the FACS Calibur. The following protocol describes the use of CaliBRITE beads and the accompanying FACSComp software (both from Becton Dickinson); alternatively, other brands of standard fluorescent beads can be used in conjunction with the CELLQuest software.
A complete description of FACSComp is found in the FACS Calibur FACSComp Software User's Guide. FACSComp is an interactive program that performs three functions: automatic adjustment of PMT voltages, automatic adjustment of FL1 and FL2 electronic compensation, and determination of instrument sensitivity with regard to FSC, SSC, FL1, and FL2 parameters.
Additional Materials (also see Basic Protocol)
CaliBRITE standard fluorescent beads (Becton Dickinson) FACSComp software program (Becton Dickinson)
1. Prepare one tube of unlabeled beads and one tube of mixed (unlabeled + FITC- + PE-labeled) beads as described in Chapter 3 of the FACSComp Software User's Guide.
Diluted beads can be stored 3 to 4 days in the dark' refrigerated' and free of microorganisms. If one or more of the FACSComp sensitivity tests fails' fresh bead samples must be prepared and reanalyzed. If the newly prepared beads also fail' BD should be contacted.
2. Launch FACSComp on the Macintosh. Enter the appropriate information in the Sign Up display and click Accept.
3. Enter the lot numbers of the unlabeled FITC- and PE-labeled CaliBRITE beads in the Set Up display. Click on Accept.
4. Insert the sample of unlabeled beads. Push the Run button.
5. Select the Start button, located at the bottom of the FACSComp window, to initiate automatic adjustment of photomultiplier tubes.
When test is complete the message PMTs Set Successfully is displayed.
6. Remove sample of unlabeled beads and replace with three-bead mixture.
7. Select Comp from the main menu or Next from the menu at the bottom for automatic fluorescence compensation adjustment.
When adjustment is complete the message Compensation Set Successfully is displayed.
8. Select the Sens button from the main menu across the top of the screen or Next from the menu in the window at the bottom. Turn on and off FACS Calibur test pulses on and off when prompted by FACSComp.
Sensitivity results will be shown and will print automatically upon quitting FACSComp if the option to do this was saved in the Preferences menu. The printout should be archived for future comparisons.
An instrument-settings file containing only the current FACSComp settings is updated after the sensitivity tests. The file is called Calib' and is stored in the BD Preferences folder' located in the Preferences folder in the Systems folder.
Immunofluorescence and Cell Sorting
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