#COMPENSATION FLOWJO HOW TO#
I'm not really sure how to solve this but hopefully someone more mathematically savvy than I am can come up with a way.įor now, I think the best way to deal with this is to use metal-minus-one or metal-minus-multiple controls for channels suffering from high spillover and use these to determine your positive/negative gates.Īdeeb: the limited testing work we've done around compensation with collaborators largely agrees with your comment about the hockey stick. So the problem with applying a standard compensation matrix is that it ends up compensating values of A where there is in fact no apparent spillover happening, hence all the negative values. This means that instead of increasing linearly as signal A goes up, there is a lower/intermediate range of channel A where these is no apparent spillover into channel B and then an uptick once you hit the limit of detection (sort of a "hockey stick"). I believe the issue is related to the fact that even if the spillover in channel B may be a fixed percentage of channel A, at lower values of A it falls below the limit of detection in B. In the case of isotopic impurity in particular, which should be fixed, I would imagine that you should be able to apply a standard matrix based on the known isotopic ratios, but when I've tried to do this the resulting increase in negative noise seems to outweigh any benefits in reducing spillover (much like what you experienced). I've tried applying compensation matrices to CyTOF data to deal with spillover, but for reasons I don't entirely understand it never seems to work particularly well. I did this to the best of my abilities, but because I'm generating a panel to study mammary tissue, I don't have (m)any ab's that are exclusive.Īll comments and help are much appreciated! I asked around about this and (if people even bother with spillover) they tell me to properly titrate and swap metals around so you don't end up with weak ab's next to strong ab's and also you should know which ab's are mutually exclusive (so you know a certain cell can never have both antibody A and antibody B on it and if you do see it, you gate it out). So my question: how should I deal with this spillover? Specifically, how do you generate a proper compensation matrix for CyTOF data?
#COMPENSATION FLOWJO PDF#
I've tried to explain it with some figures (see the PDF in the link below, sorry I didn't figure out how to easily insert pictures into this post) In order to determine the concentration of antibody to use, I have been looking at the separation of signal (low vs high) and the amount of spillover the antibodies generate.Ĭoncerning this last point, I'm not really convinced that compensation is not required for CyTOF. The detectors, or channels, in the instrument are designed to detect a very specific range of Compensation controls (single stain samples) must now belong to a compensation group inĬompensation has undergone a major redesign for FlowJo Version X! Compensation in flow cytometry is the process of correcting for fluorescence spillover emissions.ĬHAPTER 2 – Creating a FlowJo compensation matrix from single stain controls back to Compensation Workflow Overview Compensation has been reworked for version 10.Being a relatively new CyTOF user, I recently conjugated and titrated almost all my antibodies on helios. In this video, FlowJo Application Scientist Mike Stadnisky introduces you to the newest version of FlowJo with a 24-part run throuįlowJo gives you the ability to compensate your data. This may be necessary in cases where the compensation was inappropriately set during sample collection (although if the sample was over-compensated, then there is no recourse). Also, there may be casesĪccelerate your discovery with the latest features that come with FlowJo v10.6.1: Spectral compensation allows users who collect more channels than they have parameters to get cleaner data if properly done. In spectral flow, light is collected in all detectors for allįlowJo is your biggest fan and strives to be an outstanding source of support.
#COMPENSATION FLOWJO SOFTWARE#
We’re here to help you accelerate routine phenotyping, take your immunology research to the next level, and get you from data to results―one cell at a time.įlowJo applies the compensation matrix generated by acquisition software automatically upon loading data. #Flowjo 10 apply compensation matrix software# If you did not compensate on the machine or use fcs2.0 data, the parameters will not have the comp-prefix. #Flowjo 10 apply compensation matrix software#.
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