Invitrogen™ OneComp eBeads™ Compensation Beads

Catalog No.	Quantity
50-112-9041	25 tests
50-112-9031	100 tests

Catalog No. 50-112-9041 Supplier Invitrogen™ Supplier No. 01111141

Description

Spherical particles that can be stained with individual fluorochrome-conjugated antibodies for use as single-color compensation controls. OneComp eBeads react with antibodies of mouse, rat and hamster origin, and are immunoglobulin light chain-independent. 1 drop (50μL)/test

• Each drop of beads contains both a positive population that will capture any mouse, rat or hamster antibody and a negative population that will not react with antibody
• This bimodal distribution can be used for single-color compensation controls in multicolor flow cytometry experiments
• Cross-reacts to some antibodies of rabbit origin, but have not been extensively tested for this reactivity
• Designed for use in compensation with all fluorochromes excited by blue (488nm), green (532nm), yellow-green (561nm), and red (633 to 635nm) lasers
• Compatible with eFluor™ 450 but is not optimized for compensation of other fluorochromes excited by a violet (405nm) laser

Specifications

• For Use With (Equipment): Flow Cytometer
• No. of Tests: 25 tests
• Quantity: 25 tests
• Product Line: eBeads
• Product Type: Compensation Beads

Frequently Asked Questions (FAQs)

I am performing flow cytometry. My compensation matrix has values over 100. That's not possible is it?

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.

I’ve been told that I need to compensate my data for flow cytometry analysis. What does that mean?

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

What kind of controls do I need for flow cytometry?

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.

Why should I worry about compensation in flow cytometry analysis?

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.

How do I make compensation controls for antibodies?

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).

Is it necessary to wash OneComp eBeads after staining?

One wash with 2 mL of Flow Cytometry Staining Buffer (Cat. No. 00-4222-26, 00-4222-57) is recommended for ideal staining of OneComp eBeads. However, it may be possible in some cases to use unwashed OneComp eBeads, if the antibody staining concentration is low enough. In testing, it appears that concentrations starting below 0.06 µg per test may provide appropriate compensation values. Usage in this way should be confirmed in the individual antibody of interest.

OneComp eBeads are a mixture of negative and positive beads. Can I use a universal negative in auto-compensation software?

Yes. Since OneComp eBeads are a pre-mixed suspension of both negative and positive beads, there will always be a tube-specific negative population, but it is not required that the software use this population. Just include an unstained sample along with your single-color controls and be sure the gate for positive events is assigned to the positive population for each sample. The software will then use the universal negative sample and ignore the tube-specific negative populations. This should be true for any analysis software with auto-compensation features, including Flowjo and FACSDiva.

My antibody seems to stain the OneComp eBeads dimly. Are there any recommendations on how to improve the signal?

Rarely, there may be clones that are not captured well by OneComp eBeads. In such cases, it may be possible to improve the signal brightness by increasing the staining concentration, the incubation time, or both. In some cases, a different antibody that is conjugated to the same fluorochrome may be used instead.

Why do IgM antibodies stain OneComp eBeads less brightly than IgGs and will this affect my compensation values?

IgM antibodies are pentamers in solution. It is believed that these structures cause some steric hindrance resulting in less fluorochrome conjugates being captured by the beads. Compensation values should still be set appropriately using OneComp eBeads.

Can one sample tube of OneComp eBeads be used with multiple antibodies at once?

No. OneComp eBeads are designed to be used as single-color compensation controls only.

Is there flexibility in staining incubation times with OneComp eBeads?

Incubations of 5-90 minutes have been tested and may be suitable for most clones. However, deviation from the suggested 15-30 minute incubation as written in the protocol should be tested on an individual basis.

Are OneComp eBeads compatible with auto-compensation software?

Yes. After staining, OneComp eBeads provide positive and negative peaks that can be used with auto-compensation software.

Can OneComp eBeads be used with violet laser-excited fluorochromes?

Due to background autofluorescence issues, OneComp eBeads are not optimized for use with violet laser-excited fluorochromes. However, OneComp eBeads can be used with eFluor 450- and Pacific Blue-conjugated antibodies because there is no spillover into the closest detector (FITC) to compensate.

Can I use OneComp eBeads to compensate for Fixable Viability Dyes or Cell Proliferation Dyes?

No. Since these dyes are not antibodies, they are not compatible with OneComp eBeads. However, it is possible to use cells for this compensation control while using OneComp eBeads for the rest of the normal antibody-stain compensation controls.

Do OneComp eBeads work with antibodies of rabbit origin?

While OneComp eBeads were not designed with this intent, they do exhibit some reactivity to rabbit IgG. In some cases, OneComp eBeads may be useful for compensation of rabbit antibody conjugates, but this must be determined for each antibody.

What host species of antibodies do OneComp eBeads work with?

OneComp eBeads capture mouse, rat and hamster (Armenian and Syrian) antibodies of IgG and IgM classes, independent of light chain. This means OneComp eBeads are compatible with almost all direct-conjugates used in flow cytometry.

Sometimes my positive population appears as a tight doublet when using the eBioscience OneComp eBeads. What should I define or gate as positive?

There is some antibody-based variation on the shape of the positive peak. When the positive peak appears as a doublet, it is appropriate to gate on the entire positive population. Appropriate compensation values will typically result from this strategy.

Should fluorescence PMT voltages be set with eBioscience OneComp eBeads or cells?

Cells. Fluorescence-PMT voltages should be adjusted as desired for unstained cells. Stained cells and stained beads should then be checked to be sure that the voltage settings keep the positive populations on scale. If they are off scale, voltages should be adjusted until the positive populations are on scale. Once you have begun setting up compensation, only forward scatter and side scatter parameters should be adjusted to properly display beads or cells. Changes to any other voltages after compensation setup has begun will result in incorrect compensation settings.

Is it necessary to use exactly one test of antibody when staining eBioscience OneComp eBeads?

No. One test of antibody is recommended for ease of use, since it is the same quantity that would be added to cells in the experiment. However, since the brightness of the stained bead is dependent on the bead's specificity and not on the test antibody's specificity, it is not critical that the antibody be used at its optimal concentration. In general, antibodies tested well between 1.0 ug and 0.03 ug per sample. Some antibodies may need titration for optimized performance with OneComp eBeads.

For Research Use Only. Not for use in diagnostic procedures.