12/8/2023 0 Comments Compensation flowjo![]() The unused channel with the brightest signal is your best choice. A nice way to identify this channel is to drag the clean-up gate of the unstained control into the layout editor, right-click on it and choose to make a multigraph overlay of all histograms. Recommendation: When selecting a channel to designate as the AF channel, it can be useful to pick the unused channel for which AF is the highest. From the dropdown menu in the Negative column the user has the option to select to use a value of zero for the MFI of the background or ‘negative’ population.Ĭompensation will then proceed in the traditional manner by solving a system of linear equations with a target of setting the MFI of the ‘positive’ population equal to the MFI of ‘negative’ for every parameter that a compensation control tube was not stained with. The compensation wizard in FlowJo will auto-gate what will typically be a monolithic population on the clean-up gate of unstained cells, deem it the ‘positive’ population, and calculate the median fluorescent intensity (MFI) of this population in the selected channel. This is a reasonable assumption in most experiments as the instrument derived background signal is relatively invariant to signal strength 2, and thus an equal contributor to all cells regardless of type. We make the assumption that the signal in the unstained control is completely attributable to AF. SSC signal to attempt to include cells with similar AF make sure this population is the population you would like to use for AF estimation. The wizard will create a clean-up gate on a population of cells that have a consistent FSC vs. Start the wizard and assign the unstained control to a detector. To use this approach in the FlowJo Wizard, be sure to include the unstained control in the compensation group. ![]() In both approaches, AF is treated as an additional parameter to estimate during compensation, thus requiring both an empty detector and an unstained control to serve as a representative primary channel for an AF parameter and the compensation control, respectively. An estimate produced on a heterogeneous population will result in an ‘average’ AF across all included cell types, and a larger variance in over and under subtraction. If the population is homogeneous then the estimate will be more likely to subtract an appropriate amount of AF per cell. In FlowJo, there are two methods of estimating the contribution of AF per detector, and then subtracting it out: either to assume that the background signal without AF is 0, or to use AutoSpill 1 to create a linear regression based model of AF.īoth methods come with the caveat that they produce an estimate of the average AF of all the cells in the population used for the estimate. It also varies by cell depending on the size and complexity of the cell. It can contribute substantially to background noise and has the potential to obscure dimly positive populations. Methods of subtracting autofluorescence post-acquisitionĪutofluorescence (AF) is the natural emission of light by cells and other cytometric targets.
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