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HomeFlow Cytometry3-Step ColorWheel® Flow Cytometry Reagent Preparation Protocol

Simple 3-Step ColorWheel® Flow Cytometry Reagent Preparation Protocol

Through their mix-and-match ability, ColorWheel® flow cytometry antibodies and dyes are designed to provide greater flexibility in multicolor assay design and expansion than pre-conjugated primary antibodies, while offering enhanced simplicity compared to secondary conjugated antibodies and labeling kits. See just how simple it is to create your own optimal flow cytometry reagents with our 3-Step ColorWheel® Flow Cytometry Reagent Preparation protocol.

To learn more about using ColorWheel® antibodies and dyes, see our Frequently Asked Questions page.

Materials Required

This protocol requires the following materials:

  • 1 ColorWheel® antibody - sold separately
  • 1 ColorWheel® dye - sold separately
    • This is packaged with ColorWheel® finishing buffer
  • 75 µL of phosphate-buffered saline (PBS) - provided by user

You will need one ColorWheel® Antibody-Ready Dye for each ColorWheel® Dye-Ready Antibody you plan to label for this protocol along with 75 µL of PBS. The ColorWheel® finishing buffer is packaged along with each ColorWheel® Antibody-Ready Dye.

ColorWheel® Flow Cytometry Reagent Preparation Protocol

The ColorWheel® Flow Cytometry Reagent Preparation protocol takes less than 5 minutes of hands-on time for a simplified reagent preparation for flow cytometry experiments.

There are just 3 steps to create your optimal reagents:

Step 1: Spin Down Antibody, Dye, and Finishing Buffer; Resuspend

Figure 1 shows step 1 of the ColorWheel® protocol, which is:

  • Spin down the ColorWheel® dye, ColorWheel® antibody, and ColorWheel® finishing buffer.
  • Then, resuspend the dye, antibody, and finishing buffer with 25 µL of PBS each.
Spin down the ColorWheel® dye, ColorWheel® antibody, and finishing buffer and then resuspend.

Figure 1.Step 1 in the ColorWheel® Flow Cytometry Reagent Preparation protocol. Spin down the ColorWheel® dye, ColorWheel® antibody, and ColorWheel® finishing buffer and then resuspend.

Step 2: Mix Antibody and Dye

Figure 2 shows step 2 of the ColorWheel® protocol, which is:

  • Add the 25 µL of the ColorWheel® dye to the 25 µL of the ColorWheel® antibody.
  • Incubate the antibody/dye mixture at 27 °C/room temperature for 30 minutes.
Mix the ColorWheel® antibody and dye and incubate at 27°C/room temperature for 30 minutes.

Figure 2.Step 2 in the ColorWheel® Flow Cytometry Reagent Preparation protocol. Mix the ColorWheel® antibody and dye and incubate at 27 °C/room temperature for 30 minutes.

Step 3: Add Finishing Buffer and Add to Sample

Figure 3 shows step 3 of the ColorWheel® protocol, which is:

  • Add the 5 µL of ColorWheel® finishing buffer to the ColorWheel® antibody/dye mixture to create the sample-ready solution.
  • Incubate the sample-ready solution at 27 °C/room temperature for 30 minutes.
  • Add this solution to the sample.

To see how the completed reagent fits into the overall flow cytometry workflow, see our Flow Cytometry Guide.

Add finishing buffer to the ColorWheel® antibody/dye mixture and incubate at 27°C/room temperature for 30 minutes. The solution is ready to add to the sample.

Figure 3.Step 3 in the ColorWheel® Flow Cytometry Reagent Preparation protocol. Add ColorWheel® finishing buffer to the ColorWheel® antibody/dye mixture and incubate at 27 °C/room temperature for 30 minutes. The solution is ready to add to the sample.

Discover tips on finding the right fluorochrome for your experiment with our guides on selecting the right fluorochrome for your flow cytometry experiment and building the optimal panel for multicolor analysis or browse our troubleshooting guide for further advice.

Materials

Get started on creating your optimal reagents with ColorWheel® flow cytometry antibodies and dyes. You can also request more information about our trial offer for a discount on your first order.

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