"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. Data modeling was performed and displayed. The matrix is then inverted and gives the actual compensation values. Flow cytometric data were gated, compensated, and displayed using FlowJo version 3.3.3 (Tree Star, San Car- los, CA). 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. Use the bead product info sheets to determine which bead. The physical overlap between the different emission spectra of fluorochromes can activate different receptors than the ones intended for the given wavelength. Open in FlowJo, set a gate around the beads using FSC-SSC, then set a gate around each bead cluster using APC (x-axis) vs APCCy7 (y-axis). Gate successively for the lymphocytes (FSC/SSC). In this step, exclude events corresponding to cellular debris from the gate. Select population of interest by morphology (Figure 1A). Apply compensation matrix to all samples. 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. Use compensation controls acquired to create compensation matrix. FlowJo (LLC, FlowJoVx.0.7) Procedure Isolation and preparation of peritoneal cavity cells (refer to explicative video to follow steps 510). 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 controls are useful for calculating a second matrix, the spillover spreading matrix or SSM.
#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 compensation matrix is represented by the grid to the left of the sample name. 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. Create new, apply old, or compare different matrices. 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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