December 21, 2010  |  Flow Cytometry  |  By  |  0 Comments

Working with Compensation in Cytobank

Let’s just say it: compensation is a pain. Unfortunately, it’s a necessary pain if we want to accurately interpret our flow data and draw meaningful biological conclusions.

As a brief reminder of why we need to care about compensation, let’s examine the following emission spectra. We used the BD Fluorescence Spectrum Viewer; the Fluorescence SpectraViewer by Invitrogen/Life Technologies is also useful.

The plot shows the emission spectra of the commonly used fluorochromes FITC and PE, when excited by a 488 nm laser. We can measure the fluorescence signal of PE through a bandpass filter. As you can see from the plot, this filter allows light representative of the peak emission from PE to pass through (to the detector which measures this signal), but this filter also allows light from FITC to pass through. The contribution from the FITC fluorophore must be subtracted from the signal coming through the bandpass filter in order to get an accurate readout of the contribution from the PE fluorophore.

We’ll leave the in-depth discussion of compensation to the experts. In particular, we’ve found Mario Roederer’s discussions on compensation (both informal and detailed) very helpful.

We’ve created a video tutorial series on working with compensation in Cytobank, all of which are available on our YouTube channel.

Part 1 of the Compensation Tutorial Series walks you through how to use our newly released autocompensation wizard. In order to use the wizard, you will need to upload single stained bead control files along with your experimental sample files. The wizard assumes that no compensation was applied to your samples by the flow cytometer at the time of data collection. If compensation was applied on the cytometer, see part 2 of the tutorial series below. The end of the video provides a nice visual of how compensation might affect your flow data.

Part 2 of the Compensation Tutorial Series shows you how to import and modify file-internal compensation. This applies to experiments in which compensation has been calculated and applied to the FCS files on the flow cytometer. After importing the compensation matrix from your files, you can modify individual compensation values.

Part 3 of the Compensation Tutorial Series demonstrates how to import a compensation matrix from another experiment. The matrix can be imported from either a public experiment or an experiment that you have uploaded or cloned. This was also demonstrated in the PBMC Protocol Sheet Tutorial.

Part 4 of the Compensation Tutorial Series covers how to modify a saved compensation matrix. This video also provides a nice visual of how flow data might be affected by compensation.

We know working with compensation can be tricky, and we hope these tools make it a little less painful. Questions? We can help. Request support or email us at helpdesk@cytobank.org.

– Stephanie