I’ve been told that I need to compensate my data for flow cytometry analysis. What does that mean?
By running single-color controls, it is possible to remove signal of one fluorophore that spills over into the collection channel for another fluorophore.
What kind of controls do I need for flow cytometry?
For compensation, you need to prepare a singly stained sample (or compensation beads) for each color parameter that you are using. In addition, we recommend that you use FMO (flow minus one) controls. These are controls in which you label cells or beads with every color in your panel, omitting one. Make one FMO control for each color. These controls are important for helping you properly set gates on your data.
Why should I worry about compensation in flow cytometry analysis?
In a perfect world, the fluorescence emission profile for each individual fluorophore would be a very intense, narrow peak, well separated from all other emission peaks. In reality, organic dyes and fluorescent proteins have broad emission peaks, and compensation must be employed (during or after data acquisition) to correctly assign fluorescence signal to each fluorophore. Compensation is important because it removes fluorescent signal from overlapping spectra so you know that the signal you see is only the signal from the fluorophore of interest.
Identification and characterization of intestinal antigen-presenting cells involved in uptake and processing of a nontoxic recombinant chimeric mucosal immunogen based on cholera toxin using imaging flow cytometry.
Authors:Zhao W, Minderman H, Russell MW,
Journal:
PubMed ID:24197893
'Intragastric immunization with recombinant chimeric immunogen, SBR-CTA2/B, constructed from the saliva-binding region (SBR) of Streptococcus mutans antigen AgI/II and the A2/B subunits of cholera toxin (CT) induces salivary and circulating antibodies against S. mutans that protect against dental caries. We previously found that SBR-CTA2/B activated dendritic cells (DC) in the ... More
Multiparametric analysis of host response to murine cytomegalovirus in MHC class I-disparate mice reveals primacy of Dk-licensed Ly49G2+ NK cells in viral control.
Authors:Prince J, Lundgren A, Stadnisky MD, Nash WT, Beeber A, Turner SD, Brown MG,
Journal:
PubMed ID:24068668
MHC class I D(k) and Ly49G2 (G2) inhibitory receptor-expressing NK cells are essential to murine CMV (MCMV) resistance in MA/My mice. Without D(k), G2(+) NK cells in C57L mice fail to protect against MCMV infection. As a cognate ligand of G2, D(k) licenses G2(+) NK cells for effector activity. These ... More
Self MHC class I-licensed NK cells enhance adaptive CD8 T-cell viral immunity.
Authors:Stadnisky MD, Xie X, Coats ER, Bullock TN, Brown MG
Journal:Blood
PubMed ID:21436069
MHC class I (MHC I) is essential to NK- and T-cell effector and surveillance functions. However, it is unknown whether MHC I polymorphism influences adaptive immunity through NK cells. Previously, we found that MHC I D(k), a cognate ligand for the Ly49G2 inhibitory receptor, was essential to NK control of ... More