iNKT Cell Identification Protocol

Invariant natural killer T (iNKT) cells are a unique subset of T cells that secrete a wide variety of cytokines and rapidly respond to lipid antigenic stimulation [1–2]. These CD1d-restricted T cells can be uniquely identified using specific NKT cell markers. iNKT cells express CD1d T cell receptors (TCRs) along with typical T lymphocyte markers (CD3, CD4, and CD8), natural killer (NK) markers (CD161 and CD56), and the novel 6B11 monoclonal antibody (mAb) against invariant Vα24Jα18 TCR [3–5]. Mature iNKT cells can be divided into functionally distinct subsets based on CD4 and CD8 expression: CD4+ CD8-, CD4- CD8-, and CD4- CD8+ [1–3]. This T cell identification procedure helps provide a clear method for the identification of iNKT cells using these specific markers.

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• Anticoagulant for blood collection (EDTA or Heparin) (e.g., UltraPure 0.5M EDTA, pH 8.0, Cat. No. 15575020)
• Materials for standard isolation procedure for peripheral blood mononuclear cells (PBMCs)
• Dulbecco’s phosphate-buffered saline (dPBS) (e.g., Gibco PBS, pH 7.4, Cat. No. 10010023)
• 15 mL and 50 mL conical tubes (e.g., Nunc 15 mL and 50 mL conical sterile centrifuge tubes, Cat. No. 339650 or Cat. No. 339652)
• Ficoll-Paque® density separation medium (e.g., Fisher Scientific Cytiva Ficoll-Paque™ PLUS Media, Cat. No. 45-001-749)
• Fetal Bovine Serum (FBS) (e.g., Gibco Fetal Bovine Serum, Cat. No. 26140079, or Fetal Bovine Serum One Shot format, Cat. No. A3160401)
• Bovine Serum Albumin (BSA) (Optional, for preventing clogs)
• Turk or Trypan blue (for cell counting) (Gibco 0.4% Trypan Blue, Cat. No. 15250061)
• Flow cytometry staining buffer (Flow cytometry staining buffer, Cat. No. 00-4222-26)
• mAbs for identification of iNKT cells, such as TCR Vα24Jα18 (6B11), CD3, CD4, CD8, CD161

1. Collect at least 30 mL of whole blood in anticoagulant (EDTA or Heparin) and proceed with PBMC isolation within 4 hours of collection.
2. Divide the blood equally into two 50 mL conical tubes and dilute with an equal amount of dPBS (1:1).
3. Add 15 mL of Ficoll-Paque® at the bottom of two new 50 mL conical tubes.
4. Gently add 30 mL of the diluted whole blood samples over the Ficoll-Paque® while being careful to not break the Ficoll-Paque® surface.
5. Centrifuge the tubes at 400 x g for 20 minutes at room temperature with the brakes off.
6. Aspirate the upper layer while leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase.
7. Carefully harvest the mononuclear cell layers from both tubes into a new 50 mL conical tube.
8. Gently top up the new 50 mL conical tube with dPBS to wash the cells.
9. Centrifuge the cells at 400 x g for 5 minutes at 4°C. Carefully remove all supernatant and discard.
10. Repeat steps 8 and 9 to wash the cells again.
11. Resuspend the cells in an appropriate volume of dPBS to perform a cell count and viability analysis using Turk or Trypan Blue.

1. Prepare tubes for each single-stained control, fluorescence minus one (FMO) control and iNKT antibodies for identifying cells of interest.
2. Resuspend the cells to 5 x 10⁶ cells per tube.
3. Wash the cells with dPBS+0.5% FBS. Centrifuge the cells at 300 x g for 5 minutes at room temperature. Discard the supernatant.
4. Resuspend the cells with an appropriate amount of dPBS+0.5% FBS. Add the antibodies to their respective tubes.
5. Vortex the tubes and incubate them for 20 minutes at 20°C in the dark.
6. Wash the cells with dPBS+0.5% FBS. Centrifuge the tubes at 400 x g for 5 minutes at 20°C. Discard the supernatant.
7. Resuspend the cells in dPBS+0.5% FBS to a concentration between 4 x 10⁶ and 10 x 10⁶ cells/mL.
8. Acquire the cells using a flow cytometer.

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1. Hogquist, K., & Georgiev, H. (2020). Recent advances in iNKT cell development. F1000Research, 9, F1000 Faculty Rev-127.
2. Dowds, C. M., Kornell, S. C., Blumberg, R. S., & Zeissig, S. (2014). Lipid antigens in immunity. Biological chemistry, 395(1), 61–81.
3. de Jong A. (2015). Activation of human T cells by CD1 and self-lipids. Immunological reviews, 267(1), 16–29.
4. Girardi, E., & Zajonc, D. M. (2012). Molecular basis of lipid antigen presentation by CD1d and recognition by natural killer T cells. Immunological reviews, 250(1), 167–179.
5. Montoya, C. J., Pollard, D., Martinson, J., Kumari, K., Wasserfall, C., Mulder, C. B., Rugeles, M. T., Atkinson, M. A., Landay, A. L., & Wilson, S. B. (2007). Characterization of human invariant natural killer T subsets in health and disease using a novel invariant natural killer T cell-clonotypic monoclonal antibody, 6B11. Immunology, 122(1), 1–14.