- #How to compensate on flowjo 10 how to
- #How to compensate on flowjo 10 manual
- #How to compensate on flowjo 10 software
#How to compensate on flowjo 10 software
The software will now use the P3 gate to calculate compensation. The user can draw a P3 gate around the negative population.
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”.
#How to compensate on flowjo 10 manual
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”.
#How to compensate on flowjo 10 how to
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. In most research labs, there exists a notebook that contains the tried and true protocols for lab members to follow.
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