Description
Compensation is the mathematical correction for the amount of overlap (spillover) of one fluorochrome's emission spectra into another fluorochrome's channel. To calculate this overlap, record single-color stained controls for each fluorochrome in a multicolor panel. This allows the cytometer to visualize the emission of each fluorochrome in each channel, or detector, and subtract the spillover, generating a matrix to apply to multi-color experimental samples. To ensure accurate calculations, single-color controls need to be as bright, or brighter, than the marker on the cell of interest. With increased use of multi-parametric flow cytometry and the large number of fluorochromes available, it is more important than ever to ensure accurate compensation for reliable interpretation of data and reproducible experimental results.
Compensation beads are a highly desirable alternative to traditional cell-based single-color compensation. Positive beads can come pre-loaded with anti-IgG for conjugated antibody capture of mouse, rat, or hamster origin. Uncoated (negative) beads ensure standardization of the autofluorescence in each channel. Beads are preferable to cell-based compensation for several reasons:
1) No precious sample is wasted, and more sample can be used for experimental acquisition.
2) Since 100% of positive beads are able to capture the antibody, the exact experimental fluorochrome can be used regardless of a marker's cellular expression. Dimly expressed markers may not be bright enough for compensation using cells.
3) Reduced autofluorescence of beads allows for more precise calculation of spectral overlap.
Bioinformatics
| Alternate Names |
- Comp Beads
- Compensation Beads
- Compensation Capture Beads
- Flow Cytometry Compensation
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