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Be aware of possible color compensation problems running samples stained with different isomers of fluorescein! For example, the 3 plots below show Background, CD4 FITC, and CD4 Fluorescein when corrected for a FITC standard. Unstained cells appeared as shown on the left after setting up the FACSort and performing color compensation on FITC positive controls: |
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Then, both CD4 FITC and CD4 Fluorescein stained samples were run using the same instrumental settings: |
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Note that the CD4-Fluorescein emits significantly more in the FL2 (PE) channel than does CD4 FITC when the color compensation is set at 16.6% for FITC. The high voltages, of course, are the same in all 3 plots. When the CD4 Fluoresein stained cells were run, the FL2 channel required a -22.2% FL1 correction in order to bring the spillover down to background levels.
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| Take home message: Check the emission characteristics of your different "Fluorescein" reagents. The CD4 FITC is from Pharmingen and the CD4 Fluorescein from Gibco-GBL (5N6-Carboxy-Fluorescein-Succinimidyl-Ester). Both
work well - just be sure to use the correct compensation.
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