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.

How do you run compensation for flow cytometry?

How To Compensate A 4-Color Flow Cytometry Experiment Correctly

1. 4 Steps To Compensating A 4-Color Experiment.
2. Choose the correct carrier for compensation.
3. Step 2: Collect the data and make sure there is a sufficient number of events.
4. Calculate compensation correctly.
5. Apply the compensation values and inspect the results.

How do you use compensation beads?

In a 5 mL flow tube, add the same volume of your antibody as you use for your stain. Add one drop of positive comp beads and one drop of negative comp beads, or one drop of the all-in-one comp beads.

When should you run compensation controls?

Compensation is the process of correcting for spillover when one fluorophore is detected in multiple channels. It is required for most experiments of four or more colors to identify the correct signal that should be measured in each channel.

What is Flow compensation?

Pressure and temperature (PT) compensation convert a volumetric gas flow at specific conditions into an equivalent volumetric flow at base conditions. This produces a resultant value for flow that is more accurate because it is compensated for the error effects of the other variables at the operating conditions.

What is a compensation matrix?

A compensation matrix is calculated using single color fluorescent control files that are collected on the ImageStream in the absence of brightfield illumination and SSC. Once the matrix is created it can then be applied to the experiment data when batch processing or opening a raw image file.

Can you use FITC and PE together?

Relative contribution. In some experiments FITC may be combined with other dyes, for example PE, that emit yellow and orange photons. In those cases the relative contribution of each fluorophore to the signal in a given detector must be determined (Figure 11).

What is compensation matrix?

How do you stain comp beads?

Incubate at 2-8°C for 15-30 minutes in the dark. Add 2 mL of Flow Cytometry Staining Buffer to each tube and centrifuge at 400-600 x g for 3-5 minutes. Decant supernatant and add 0.2-0.4 mL of Flow Cytometry Staining Buffer to each tube. Mix briefly by flicking or pulse vortexing before analysis.

What is FITC and PE in flow cytometry?

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™.

Why do we use pressure and temperature compensation?

Why do you do process temperature and pressure flow compensation? In steam or gas flow measurement, the density of the steam or gas changes as pressure and temperature change. This change in density can affect the accuracy of the measured flow rate if it is uncompensated. Pressure.

What does compensation mean 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.

How can I improve the performance of my flow cytometer?

We always recommend reviewing the flow cytometer manufacturer’s instructions for detailed compensation guidelines. Ensure that the cytometer is performing within specifications using standard beads. Set voltages for fluorescence channels using an unstained sample.

How to correct for spillover of fluorochromes in Multicolor flow cytometry?

During the early years of multicolor flow cytometry, where most researchers were using 2-4 fluorochromes, manual compensation was the vogue method to correct for the spillover of a fluorochrome into secondary detectors.

What is fluorescence compensation and how is it used?

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.