Do PE and APC need compensation?
For APC, the excitation source can vary depending on the available lasers. The 594 nm or 633 nm laser line are suitable. For emission filters, the Cy5 filter (660/40) is recommended. For multicolor analysis, compensation may be required before analyzing data.
What is PE and FITC?
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. The FITC/PE Compensation Standard is a mixture of 4 populations of microspheres: FITC, PE, FITC/PE, and AutoFluor™.
How does compensation work 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.
Can I use PE and APC 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.
What color is FITC?
In the case of FITC, its maximum absorbance falls within the blue spectrum. Therefore, the blue 488 nm laser, which is close to FITC’s absorbance peak of 490 nm, is commonly used to excite this fluorophore. FITC emits fluorescence from 475 to 650 nm, peaking at 525 nm, which falls in the green spectrum.
Can I use APC and PE together?
Can you use APC and PE Cy5 together?
For example, PE-Cy5 is very bright with regard to stain index (Table 1); but it has considerable spillover into the APC detector. While these two fluorochromes can be used together, the resolution sensitivity in APC will be reduced compared to, for example, a combination of PerCP-Cy5.
What color is PE?
Phycoerythrin (PE) is a red protein-pigment complex from the light-harvesting phycobiliprotein family, present in red algae and cryptophytes, accessory to the main chlorophyll pigments responsible for photosynthesis.
Why does flow cytometry require compensation?
Compensation is necessary in order to be able to differentiate between populations of cells. The matrix is then inverted and gives the actual compensation values. The flow cytometer then uses these values to correct the overlap in each detector for each colour.
Why is compensation used in flow cytometry?
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.