Channel Calibration

Quantitative flow cytometry is used to determine the actual number of fluorescent ligands (fluorochromes or fluorochrome-protein conjugate molecules) labeling each particle. The technique requires calibration of the fluorescence channel number with standards of known fluorochrome content. The standards define the relationship between the observed fluorescence level and the number of bound fluorochrome molecules.

 

Several companies have specially designed beads of different intensities where the precise number of dye molecules per bead is known. By establishing a standard curve correlating the channel number to the number of dye molecules per bead, it is possible to determine the absolute number of molecules per cell of antigen, antibody, and other useful markers.

 

This type of calibration has traditionally been a very labor intensive process, requiring you to use your flow cytometry software to obtain a set of statistics from the beads and the sample, then entering the statistics into a spreadsheet, creating a calibration curve, determining the standard channel values, and finally performing more calculations using the spreadsheet to convert the statistics to the number of binding sites per cell.

 

FCS Express can assist in automating the entire process for you using a weighted linear regression. Once you have set up a channel calibration, all plots will automatically display the calibrated axes if desired, and the statistics will be displayed as calibrated values. Tokens may be used to monitor if a calibration has been applied to an axis, what the last channel calibration loaded in the layout was, and provide detailed information about channel calibrations applied in the layout or to a specified plot. To set up a channel calibration, you must have a set of beads for which the number of fluorochrome molecules is precisely known. In addition, you must know the effective fluorescence-protein ratio to determine the number of dye molecules attached to each marker on the cells.