#Compensation in flowjo software#

Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Here, we present AutoSpill, an alternative method for calculating spillover coefficients. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Even the advent of spectral cytometry cannot circumvent the spillover problem, with spectral unmixing an intrinsic part of such systems. PMID 330738.Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. The Journal of Histochemistry and Cytochemistry. 'Two-color immunofluorescence using a fluorescence-activated cell sorter'. 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. This is done by measuring the spectral overlap of the different fluorochromes and using the measured values to create a matrix. 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. during a two colour experiment, where mouse splenocytes were stained with fluorescein and rhodamine. The first data compensation was done in 1977 by Michael Loken et al.

#Compensation in 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.