The RayBio^® Human TBNK Flow Cytometry Kit is designed to determine the percentages of mature human lymphocyte subsets in peripheral whole blood (WB). The subsets include T lymphocytes (CD3⁺), Helper T lymphocytes (CD3⁺CD4⁺), Cytotoxic T lymphocytes (CD3⁺CD8⁺), B lymphocytes (CD19⁺) and Natural killer (NK) lymphocytes (CD3^-CD16&56⁺). Our kit contains individually packed fluorochrome-labeled antibodies, antibody diluent, fluorescent counting beads for absolute number counts (cells/μL) of gated cell populations, and lysing solution to minimize the red blood cells. This kit is easy and convenient to use, and a perfect tool for immunophenotyping peripheral lymphocytes of human whole blood.

• RayBright^® Violet 450 Anti-human CD19
• RayBright^® Violet 500 Anti-human CD8
• RayBright^® Blue 488 Anti-human CD3
• PE Anti-human CD4
• PerCP Anti-human CD45
• APC Anti-human CD16
• APC Anti-human CD56
• Antibody Diluent
• RayBio^® 10X Whole Blood Lysing Solution

• Flow cytometer with violet, blue and red lasers
• 1.5 mL polypropylene microcentrifuge tubes or similar
• 12 × 75-mm tubes or similar
• De-ionized (DI) H₂O
• Centrifuge
• Vortex mixer
• Optional: Fluorescent Counting Beads

1. Dilute the RayBio^® 10X Whole Blood Lysing Solution 10-fold with de-ionized (DI) H₂O to prepare 1X lysing solution.
2. Prepare the antibody cocktail in a 1.5 mL tube. For each test, add 30 μL of the Antibody Diluent and 2.5 μL of each antibody reagent. The final volume of antibody cocktail should be 50 μL per test.
3. Label each 12 × 75-mm tube. Add 50 μL of well-mixed anticoagulated whole blood from each sample to the bottom of the appropriate tube.
4. Add 50 μL of antibody cocktail to the bottom of each tube and vortex gently to mix.
5. Incubate for 20 minutes at room temperature, protected from light.
6. Add 1 mL of 1X lysing solution to each tube and vortex gently to mix.
7. Incubate for 20 minutes at room temperature (20°C–25°C), protected from light.
8. Centrifuge the samples at 500 x g for 5 minutes. Carefully discard the supernatant without disturbing the cell pellet.
9. Resuspend the pellet in 0.5 mL of 1X lysing solution. Samples are now ready to be analyzed on a flow cytometer.

Store kit at 4°C, protected from light for up to three months for full functionality.