OneComp eBeads™ Compensation Beads, 100 tests - FAQs

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.

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

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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.

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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.

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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).

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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.

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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.

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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.

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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.

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

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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.

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

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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