AbC™ Total Antibody Compensation Bead Kit, 25 tests - FAQs

Here are some tips to follow:
- Make sure that the beads were never frozen.
- Avoid mixing by vortexing too long (no longer than 10 seconds) or sonication as this may promote denaturation of the protein on the surface of the beads.
- Use the product within the warranty period (one year).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Yes, the AbC Total Antibody Compensation Bead Kit can bind Fab dimers as long as the Fab dimers are derived from either mouse, rat, hamster, or rabbit species.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

As an alternative, you may covalently attach your antibody to colorless, 5 µm Aldehyde/Sulfate Latex Beads (Cat. No. A37306). The aldehyde moiety on the bead surface directly binds with primary amines on the surface of the antibodies in simple buffers such as PBS (pH 7.4). Make sure that the buffer does not contain any primary amines (i.e., do not use Tris or glycine buffers). To avoid making bead-antibody-bead-antibody multimers, add excess antibody relative to the beads.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

It is possible. But if you have a lot of values over 100, you probably need to look at the voltages that you are using for your data collection.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

By running single-color controls, it is possible to remove signal of one fluorophore that spills over into the collection channel for another fluorophore.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

For compensation, you need to prepare a singly stained sample (or compensation beads) for each color parameter that you are using. In addition, we recommend that you use FMO (flow minus one) controls. These are controls in which you label cells or beads with every color in your panel, omitting one. Make one FMO control for each color. These controls are important for helping you properly set gates on your data.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

In a perfect world, the fluorescence emission profile for each individual fluorophore would be a very intense, narrow peak, well separated from all other emission peaks. In reality, organic dyes and fluorescent proteins have broad emission peaks, and compensation must be employed (during or after data acquisition) to correctly assign fluorescence signal to each fluorophore. Compensation is important because it removes fluorescent signal from overlapping spectra so you know that the signal you see is only the signal from the fluorophore of interest.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

All that you need for compensation controls is an unstained sample of your cells (or negative control beads) and samples with each of your fluorophores - one per tube. You want the single-stained controls to be as bright as you expect your brightest sample to be. Antibodies can be bound to beads instead of cells using our AbC Total Antibody Compensation Bead Kit (Cat. Nos. A10513 and A10497).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.