CD1d is a membrane protein non-covalently bonded to β2-microglobulin (β2m) and shows high homology between human and mice. CD1d can present α-galactosylceramide (α-GalCer), a glycolipid extracted and isolated from the marine sponge, and this complex can activate human and murine CD1drestricted NKT cells. CD1d Tetramer-PE is a reagent prepared by tetramerization of complexes of CD1d and β2m by PE- or APC- labeled streptavidin. Binding this reagent to α-GalCer enables highly sensitive detection of CD1d-restricted NKT cells and can be combined with antibodies to study NKT cell function by flow cytometry.

Reference:
1) Stephane S et al. J. Immunol. Methods 268, 107−121 (2002)
2) Matsuda JL et al. J. Exp. Med. 192, 741−754 (2000)
3) Benlagha K et al. J. Exp. Med. 191, 1895−1903 (2000)

PBMC were separated from peripheral blood of healthy subjects and incubated at room temperature for 5 minutes with 10 µL of Clear Back (Human Fc receptor blocking reagent, Code No. MTG-001). CD3-FITC and human CD1d Tetramer-PE (with or without binding of α-GalCer) were added and incubated for 30 minutes at 4°C protected from light, and cells were analyzed by flow cytometry. Results showed total cells contained 0.01-0.55% NKT cells, as defined by CD1d/CD3 dual positivity.

PBMC were separated from peripheral blood of healthy subjects and incubated at room temperature for 5 minutes with 10 µL of Clear Back (Human Fc receptor blocking reagent, Code No. MTG-001). Human CD1d Tetramer-PE (with or without binding of α-GalCer), anti-human TCR Vα24-Jα18 (clone 6B11)-FITC, and mouse IgG1-FITC (isotype control) or CD3-FITC, were added and incubated for 30 minutes at 4°C protected from light, and cells were analyzed by flow cytometry. Results showed total cells contained 0.47% NKT cells as defined by CD1d/CD3 dual positivity. On the other hand, 0.29% NKT cells as defined by CD1d/TCR Vα24-Jα18 dual positivity.

C57BL/6 mouse splenocytes were stained with CD4-FITC and mouse CD1d Tetramer-PE (with or without binding of α-GalCer) for 30 minutes at 4°C protected from light. Measurement by flow cytometry resulted in detection of NKT cells corresponding to 2.33% of all the cells.

Dissolving and loading of α-GalCer used with CD1d Tetramer

★α-GalCer is not loaded on Code No. TS-HCD-1, TS-HCD-2, TS-MCD-1 and TS-MCD-2.

T-Select CD1d Tetramer Product list