![]() After acquiring each compensation control, after gating on the FCSxSSC plot (P1), the histogram in question will have a P2 gate that is placed around the positive population. As shown in the figure below, it is clear that a single, unstained sample cannot be used to properly set the background.įigure 1: Unstained cells (red) and beads (blue) have different background fluorescence.įor those using BD DIVA software to acquire samples when setting up compensation, make sure to uncheck “Include separate unstained control tube/well”. This was the default method when performing manual compensation.Īs discussed previously, the Universal Negative violates the 2 nd rule of Compensation, which states the positive and negative carrier must have the same background.Ī lot of the automated analysis packages on flow cytometry software, both acquisition and analysis, offer the ability to identify a single sample that is supposed to be representative of the background fluorescence of the population. The idea behind the Universal Negative is that a single tube, typically unstained cells, is used to set the negative population for establishing the compensation matrix. This blog article will help shine light on some of these historical practices and why they need to be changed. The last 2 blog articles have discussed the theory and practice of compensation. It is for this reason that there are suboptimal practices that permeate flow cytometry experiments to this day. The “truths” in this book are not always right anymore, but the new user doesn’t necessarily know any differently. Unfortunately, these pages are not refreshed with the best practices that have evolved over time as the technology and our understanding has changed and grown. These protocols are time-honored and tested, so the new researcher doesn’t question the wisdom of the “Protocols Book”. These hallowed, often coffee-stained, pages teach the researchers everything - from how to make media, passage cells, and run restriction digestions, to how to prepare cells for flow cytometry analysis. A compensation group for each unique compensation matrix is necessary since only one compensation matrix can be created per compensation group.In most research labs, there exists a notebook that contains the tried and true protocols for lab members to follow. (Simply create a new group and assign it the role of ‘Compensation’). If multiple matrices are required, several compensation groups must be created. Once you have a matrix in FlowJo, it will appear as a node similar to other nodes in FlowJo (gates, stats, platforms, etc.): In addition to automatically organizing your single-stain controls, the compensation group also stores your matrix nodes. ![]() You can set default inclusion rules in Compensation Preferences. You can see the default inclusion criteria for the Compensation group by double-clicking it:īy default, we’ll include samples where any keywords in the samples’ metadata contains the phrases of either “comp” or “unstained”. Probably the first thing you’ll notice about the Workspace window changes is the new special Group “Compensation” :įlowJo scans your files as you load them into the workspace, and if any of them match our criteria for compensation controls, they will be added to this group. ![]()
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