This brief guide is to help users new to compensation calculations and experienced flow-maestros alike breeze through this process in a painless and professional fashion. The Journal of Histochemistry and Cytochemistry. The Compensation Wizard in FlowJo is one of the most frequently used platforms, and by extension potentially the greatest source of confusion on a per-cytometrist basis. The grid badge indicates the compensation status of your samples in the workspace. "Two-color immunofluorescence using a fluorescence-activated cell sorter". The FlowJo workspace adorns samples with new icons, presented in three columns known as badges. Annals of the New York Academy of Sciences. "Fluorescence spectral overlap compensation for any number of flow cytometry parameters". "Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats". The flow cytometer then uses these values to correct the overlap in each detector for each colour. The matrix is then inverted and gives the actual compensation values. Automated compensation was calculated by. This is done by measuring the spectral overlap of the different fluorochromes and using the measured values to create a matrix. Compensation Beads (Thermo Fisher) were used according to manufacturer's. This correction is called compensation.Ĭompensation is necessary in order to be able to differentiate between populations of cells.
The ability to correct this stems from the fact, that the overlap is a linear function, so the measured signal can be averaged and thus corrected. The physical overlap between the different emission spectra of fluorochromes can activate different receptors than the ones intended for the given wavelength. When one cell is marked by two or more fluorochromes, the added brightness of one fluorochrome to the other creates significant background noise and affects the strength of the signal. Proper compensation requires that the positive and negative populations be gated on similar cell types (i.e., that have the same autofluorescence when unstained). If compensation is incorrect, it is simple to generate a new compensation matrix to apply to the samples. However if clogging is a recurring problem, you may consider filtering your samples prior to running them. during a two colour experiment, where mouse splenocytes were stained with fluorescein and rhodamine. First of all, we will open the first sample (FITC Comp sample), in order to make a lymphocyte gate. If you are using FlowJo, you can use the export function to create new fcs files where all slashes are replaced with underscores. The first data compensation was done in 1977 by Michael Loken et al.
#Compensation flowjo software#
The compensation can be done through different flow cytometry software such as Flowjo, Flowlogic, Kaluza etc.
This creates a signal overlap (spillover) which cannot be removed by the optical system and has to be corrected electronically. The photons emitted by fluorochromes have different energies and wavelengths and as flow cytometers use photomultiplier tubes (PMT) in order to convert the photons into electrons, the detector can register the signal from more than one fluorochrome. In cytometry, compensation is a mathematical correction of a signal overlap between the channels of the emission spectra of different fluorochromes.
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