ColorWheel^® Flow Cytometry Protocol

The following protocol is a guideline for flow cytometry analysis using a ColorWheel^® antibody with a ColorWheel^® dye. Both are required to make the ColorWheel^® antibody-dye solution that may be used in your flow cytometry protocol.

• Materials
• Antibody Preparation Protocol
• PBMC Sample Preparation Protocol
• Cell Surface Staining Protocol
• Intracellular (Cytoplasmic) Staining Protocol
• Related ColorWheel^® Products

Materials

• ColorWheel^® Dye Set (ColorWheel^® Finishing Buffer included)
• ColorWheel^® Antibody
• DPBS: Product No. D8537 or D1408 (10X Concentrate)
• RPMI 1640 Media: Product No. R0883-500ML
• Fetal Bovine Serum (DPBS): Product No. TMS-013-B
• GlutaMAX™ Supplement: ThermoFisher Cat. No. 35050079
• Penicillin-Streptomycin (100X): Product No. P4333
• Frozen PBMC Vial: IQ Biosciences Cat. No. IQB-PMBC102
• Bovine Serum Albumin (BSA): Product No. A3059
• Sodium Azide: Product No. 71289
• 0.5 M Ethylenediaminetetraacetic Acid (EDTA): Product No. 03690
• FACS Buffer (5% BSA, 2 mM EDTA, 0.1% Sodium Azide in DPBS)
• Human Fc Blocking Solution: BD Biosciences Cat. No. 564219
• 16% Formaldehyde: ThermoFisher Cat. No. 28908
• 10X Permeabilization Buffer (0.1% Saponin and 0.09% Sodium Azide in DPBS): ThermoFisher Cat. No. 00-8333-56

Explore tips on choosing 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.

Antibody Preparation Protocol

1. Reconstitute ColorWheel^® Dye with 25 µL of DPBS.
2. Reconstitute ColorWheel^® Antibody with 25 µL DPBS.
3. Reconstitute ColorWheel^® Finishing Buffer with 25 µL DPBS.
4. Add 25 µL of ColorWheel^® Dye (Step 1) to 25 µl of ColorWheel^® Antibody (Step 2) to create 50 µL of ColorWheel^® Antibody + Dye Solution. Label the tube well.
5. Incubate ColorWheel^® Antibody + Dye Solution in the dark for 30 minutes at room temperature.
6. Add 5 µL of ColorWheel^® Finishing Buffer (Step 3) to 50 µL of ColorWheel^® Antibody + Dye Solution (Step 4) to create 55 µL of ColorWheel^® Sample-Ready Solution.
7. Incubate ColorWheel^® Sample-Ready Solution for 30 minutes at room temperature.
8. Use ColorWheel^® Sample-Ready Solution immediately or store at 4 °C until use.

For more information, see our easy 3-Step ColorWheel^® Reagent Preparation Protocol.

PBMC Sample Preparation Protocol

1. Warm up RPMI 1640 complete media (with 10% FBS, 2 mM GlutaMAX™ Supplement, 100 units/mL of Penicillin, and 100 µg/mL of Streptomycin) in a 37 °C water bath.
2. Access a frozen PBMC vial and quickly thaw cells in a 37 °C water bath.
3. Transfer cell solution to a 50 mL conical centrifuge tube.
4. Add 9 mL of RPMI complete media drop by drop.
5. Centrifuge at 400 x g for 5 minutes.
6. Discard supernatant and wash cells with 10 mL of DPBS.
7. Centrifuge at 400 x g for 5 minutes.
8. Discard supernatant and resuspend cells at 1 x 10⁶ cells/mL with FACS buffer (5% BSA, 2 mM EDTA, 0.1% Sodium Azide in DPBS).

Cell Surface Staining Protocol

1. Aliquot 1 x 10⁶ cells to each tube.
2. Block non-specific Fc receptors on cell surface with appropriate Fc blocking solution.
3. Incubate at room temperature for 10 minutes.
4. To stain cells, add 5 µL of ColorWheel^® Sample-Ready Solution per 1 x 10⁶ cells.
5. Incubate at 4 °C in the dark for at least 30 minutes.
6. Centrifuge cells at 400 x g for 5 minutes.
7. Discard supernatant and wash cells with 2 mL of DPBS per tube.
8. Repeat steps 6-7 for two additional times.
9. Centrifuge cells at 400 x g for 5 minutes.
10. Discard supernatant and resuspend cells with 2 mL of FACS buffer.
11. Analyze samples by flow cytometry.

Browse our flow cytometry troubleshooting guide for further advice on cell staining.

Intracellular (Cytoplasmic) Staining Protocol

1. Aliquot 1 x 10⁶ cells to each tube.
2. Centrifuge cells at 400 x g for 5 minutes.
3. Discard supernatant and fix cells with 1 mL of 2-4% formaldehyde in DPBS for 10 minutes at room temperature.
4. Centrifuge cells at 400 x g for 5 minutes.
5. Discard supernatant and wash cells with 2 mL of FACS buffer per tube.
6. Repeat steps 4-5 one more time.
7. Centrifuge cells at 400 x g for 5 minutes.
8. Discard supernatant and resuspend cells with 1 mL of permeabilization buffer (0.1% Saponin and 0.09% Sodium Azide in DPBS) with 5% BSA and appropriate Fc blocking solution.
9. Incubate at room temperature for 30 minutes to block cells.
10. To stain cells, add 5 µL of ColorWheel^® Sample-Ready Solution per 1 x 10⁶ cells.
11. Incubate at 4 °C in the dark for at least 30 minutes.
12. Centrifuge cells at 400 x g for 5 minutes.
13. Discard supernatant and wash cells with 2 mL of permeabilization buffer per tube.
14. Repeat steps 12-13 for two additional times.
15. Centrifuge cells at 400 x g for 5 minutes.
16. Discard supernatant and resuspend cells with 2 mL of FACS buffer.
17. Analyze samples by flow cytometry.

Have questions about ColorWheel^® products? Check out our ColorWheel^® FAQs for answers to common questions.

Related ColorWheel^® Products

ColorWheel^® technology requires BOTH a ColorWheel^® antibody AND a ColorWheel^® dye for flow cytometry research applications, such as multicolor analysis. The antibody and dye are sold separately. For a discount on your first order, you can request more information about our trial offer.