How do you compensate flow cytometry data?
How do you compensate flow cytometry data?
How To Compensate A 4-Color Flow Cytometry Experiment Correctly
- 4 Steps To Compensating A 4-Color Experiment.
- Choose the correct carrier for compensation.
- Step 2: Collect the data and make sure there is a sufficient number of events.
- Calculate compensation correctly.
- Apply the compensation values and inspect the results.
What is compensation in flow cytometry?
The term “compensation,” as it applies to flow cytometric analysis, refers to the process of correcting for fluorescence spillover, i.e., removing the signal of any given fluorochrome from all detectors except the one devoted to measuring that dye.
Why is compensation needed in flow cytometry?
Compensation is required for a flow cytometry experiment because of the physics of fluorescence. A fluorochrome is excited, and emits a photon in a range of wavelengths. Some of those photons spill into a second detector, causing single stained samples to appear double positive.
Why do we need compensation for multiple parameter fluorescence?
However, when emission spectra overlap, fluorescence from more than one fluorochrome may be detected. To correct for this spectral overlap, a process of fluorescence compensation is used. This ensures that the fluorescence detected in a particular detector derives from the fluorochrome that is being measured.
Do FITC and PE need compensation?
Why do we need compensation? where FITC bleeds into the PE channel and PE bleeds back into FITC. To correct for spectral overlap during multicolor flow cytometry experiments, color compensation must be performed. The goal of color compensation is to correctly quantify each dye with which a particular cell is labeled.
What is compensation matrix?
A compensation matrix is calculated using single color fluorescent control files that are collected on the ImageStream or FlowSight with all channels collected and in the absence of brightfield illumination or SSC.
How do you compensate with FlowJo?
1) To initiate creating a new compensation matrix in FlowJo, select the compensation group in the workspace and go to the Workspace ribbon. 2) Click the compensation icon in the Cytometry band of the Tools tab. 3) The compensation interface will launch.
Does PE bleed into FITC?
where FITC bleeds into the PE channel and PE bleeds back into FITC. To correct for spectral overlap during multicolor flow cytometry experiments, color compensation must be performed. The goal of color compensation is to correctly quantify each dye with which a particular cell is labeled.
Does PE and FITC overlap?
The FITC / PE Compensation Standard is to be used in conjunction with hardware or software to remove spectral overlap from fluorochromes into secondary fluorescence detectors of a flow cytometer.
Can you use PE Cy7 and APC Cy7 together?
In my experience, it is not advisable to use APC/Cy7 and PE/Cy7 together due to heavy cross-beam contamination as their emission max are exactly same. However, they can be used together if you are using machines like the Cytek Aurora.
