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HomeFlow CytometryColorWheel® Flow Cytometry Antibodies and Dyes: Frequently Asked Questions

ColorWheel® Flow Cytometry Antibodies and Dyes: Frequently Asked Questions


Can I pair an antibody from another vendor to a ColorWheel® dye?

No. ColorWheel® antibodies have a proprietary single-stranded DNA (ssDNA) oligo linker that is complementary with the ssDNA oligo linker on ColorWheel® dyes. Without these linkers, hybridization will not occur.

Do you just sell the oligo linker?

We currently do not sell the oligo linker as a stand-alone product.

Can you use a labeled secondary antibody with unlabeled ColorWheel® antibodies?

You should be able to use a host species compatible secondary antibody for indirect detection of ColorWheel® antibodies. However, as there are many variables present, this is not a validated approach and is not backed by our Antibody Guarantee.

Will there be cross-labeling between multiple ColorWheel® antibodies and dyes in a sample?

By following our 3-step ColorWheel® reagent preparation protocol prior to starting a flow cytometry experiment, cross-labeling should not occur. ColorWheel® finishing buffer quenches hybridization and helps to ensure each ColorWheel® dye is labeled to the intended ColorWheel® antibody.

Can a sample for flow cytometry be stained with both ColorWheel® reagents and non-ColorWheel® reagents?

Yes. ColorWheel® products are meant to be easily inserted into your existing flow cytometry assays. ColorWheel® labeled antibodies can be used simultaneously in a sample that is stained with non-ColorWheel® antibodies. For a comprehensive guide to using ColorWheel® products in flow cytometry, see our ColorWheel® Flow Cytometry Protocol.

What dye should I use for my antibody?

In general, one should pair a highly expressing antigen with dim dyes, and low expression antigens with bright dyes. For more tips, check out our guide on how to select dyes for flow cytometry.

Can I label one antibody with two different dyes?

No. ColorWheel® dyes are designed for labeling antibodies with one dye. All dyes utilize the same chemical groups on the antibody, thus we cannot guarantee the success of this type of experiment.

How much volume should I use to resuspend ColorWheel® reagents?

Our ColorWheel® reagents have been engineered to be reconstituted at 25 µL volumes of phosphate-buffered saline (PBS). We strongly recommend that all reagents be resuspended at the same 25 µL volume to ensure optimal functionality.

Refer to our simple 3-step ColorWheel® reagent preparation protocol here.

How much volume of ColorWheel® dye do I mix with ColorWheel® antibody?

After resuspending with 25 µL of PBS, we strongly recommend that you mix ColorWheel® antibodies and dyes in a 1:1 ratio. For example, mix 25 µL of dye with 25 µL of antibody. A 1:1 ratio is the optimized ratio of antibody:dye.

Refer to our simple 3-step ColorWheel® reagent preparation protocol here.

How much finishing buffer do I add to my antibody-dye solution?

We recommend adding 5 µL of finishing buffer to 50 µL of antibody-dye solution.

Refer to our simple 3-step ColorWheel® reagent preparation protocol here.

How much sample-ready solution do I add to my sample?

We recommend users initially add 5 µL of the sample-ready solution to 1 million cells. Users must empirically optimize per experiment.

Refer to our simple 3-step ColorWheel® reagent preparation protocol here.

Do I need to purify my antibody?

No. ColorWheel® antibodies and dyes are purified. They are provided as lyophilized, ready-to-assemble, and free of preservatives, carrier proteins, and biocides.

Refer to our simple 3-step ColorWheel® reagent preparation protocol here.

At what temperature should I perform the 3-step preparation protocol?

ColorWheel® antibodies and dyes can be resuspended, mixed, and incubated at 27°C/room temperature.

What is the shelf life of lyophilized ColorWheel® reagents?

ColorWheel® antibodies, dyes, and finishing buffer are all lyophilized and have undergone accelerated stability testing. The result of this testing suggests lyophilized ColorWheel® reagents are stable for at least 5 years.

How should I store lyophilized ColorWheel® reagents?

It is recommended that all lyophilized ColorWheel® reagents be stored between 2-8oC once received, but they are stable during shipment at ambient temperature. They should be protected from light and never frozen.

How should I store hydrated ColorWheel® reagents?

Once reconstituted, all ColorWheel® reagents should be stored between 2-8oC. They should be protected from light and never frozen.

What buffers are suitable for ColorWheel® reagents?

PBS buffer is suitable for the reconstitution of all ColorWheel® reagents.

Do you offer custom services?

We currently do not offer custom services.

Materials

Get started on creating your optimal reagents with ColorWheel® flow cytometry antibodies and dyes.

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