αgalcer

Manufactured by ProImmune

Sourced in United Kingdom

αGalCer is a synthetic glycolipid compound used in laboratory research. It serves as an agonist for the CD1d receptor, which is involved in the activation of natural killer T cells. This compound is primarily utilized in immunological studies to investigate the role of natural killer T cell activation in various biological processes.

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PE-conjugated αGalCer-CD1d Tetramer (1 citations)

4 protocols using αgalcer

Multi-parameter Flow Cytometry Profiling

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Six-color staining was conducted using combinations of following monoclonal antibodies: F4/80 (Serotec, Raleigh, NC, USA); CD4, CD11b (both eBioscience, San Diego, CA, USA); CD45, Gr1/Ly6C, Ly6G, CD19, NK1.1, CD8 and CD3 (all BD); CD1d tetramer loaded with αGalCer (ProImmune, Oxford, UK). Dead cells were excluded by Hoechst 33258 dye (Sigma-Aldrich, St. Louis, USA). Flow cytometric analysis was performed on a FACS-Canto (BD) and analysed with FlowJo (Tree Star, Ashland, OR, USA).

Wehr A., Baeck C., Ulmer F., Gassler N., Hittatiya K., Luedde T., Neumann U.P., Trautwein C, & Tacke F. (2014). Pharmacological Inhibition of the Chemokine CXCL16 Diminishes Liver Macrophage Infiltration and Steatohepatitis in Chronic Hepatic Injury. PLoS ONE, 9(11), e112327.

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NKT Cell Detection Protocol

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Anti-mouse αGalCer:CD1d complex antibody (L363, eBioscience, San Diego, CA)17 (link) was used to detect glycolipid antigen presentation on CD1d. CD1d-tetramer loaded with αGalCer was purchased from ProImmune (ProImmune, Oxford, U.K.), unless otherwise specified, and NKT cells were identified as CD19⁻CD3⁺αGalCer/CD1d tetramer⁺ cells. Other antibodies used for staining were CD49b (HM ALPHA2), TCR-β (H57-597), CD11c (HL3), CD44 (IM7), CD80 (16-10A1), CD86 (GL1), IgG1 (A85-1), and I-Ad/I-Ed (2G9) were all purchased from BD Biosciences; CD3 (17A2), CD19 (6D5), TNF-α (MP6-XT22) and CD69 (H1.2F3) were from Biolegend; F4/80 (BM8), IFN-γ (XMG1.2), IgG2a (eBM2a) and IgG1 (eBRG1) were obtained from eBioscience. Data were acquired on a FACSCanto™ II flow cytometer (BD Biosciences) and analyzed with FlowJo (Treestar) or BD FACS Diva (BD Biosciences) software. The details of intracellular staining see the Supplementary Information.

Wei Y., Zeng B., Chen J., Cui G., Lu C., Wu W., Yang J., Wei H., Xue R., Bai L., Chen Z., Li L., Iwabuchi K., Uede T., Van Kaer L, & Diao H. (2016). Enterogenous bacterial glycolipids are required for the generation of natural killer T cells mediated liver injury. Scientific Reports, 6, 36365.

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Quantifying NKT Cells and Cytokines

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All samples from individual subjects were tested simultaneously on blinded samples. For NKT measurements, ten ml of heparinized peripheral blood were collected before and 24 hours following each treatment, and at day 56 (end of study). PBMCs were isolated by Ficoll density gradient centrifugation and cryopreserved in liquid nitrogen until use. For staining, 1×10⁶ PBMCs were incubated with α-GalCer or vehicle loaded CD1d-tetramers (ProImmune, UK) for 30 min at 4°C, then with anti-CD3 antibody (Becton–Dickinson Biosciences) for 30 min at 4 °C. Analysis was performed on a FACSCanto using CELLQuest software (BD Biosciences).
Cytokine was measured before, 4 and 24h after the first and second injections and at the end of study using a multiplex electrochemiluminescense assay according to the manufacturer’s instructions (MSD) and analyzed using the Discovery Workbench MSD software.

Tefit J.N., Crabé S., Orlandini B., Nell H., Bendelac A., Deng S., Savage P.B., Teyton L, & Serra V. (2014). Efficacy of ABX196, a new NKT agonist, in prophylactic human vaccination. Vaccine, 32(46), 6138-6145.

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Flow Cytometric Analysis of Immune Cell Markers

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Cell surface markers were measured by a flow cytometer (FACScalibur and FACScan; BD Biosciences, San Diego, CA) using the respective monoclonal antibodies. The dead cells were excluded by forward scatter, side scatter, and propidium iodide gating. The Cytofix/CytoPerm Kit (BD Biosciences) and PerFix-pKit (Beckman Coulter) were used for the intracellular staining. The antibodies used were FITC-conjugated anti-CD3, FITC-conjugated anti-CD24, allophycocyanin-conjugated anti-NK1.1, PE-conjugated anti-IL-4, PE-conjugated anti-IFN-γ, PE-conjugated anti-LFA-1, PerCP-Cy5.5-conjugated anti-CD44, and their isotype-matched control antibodies (eBioscience), as well as PE-conjugated CD1d-tetramer preloaded with α-GalCer (ProImmune Ltd., Oxford, UK) and PE-conjugated anti-pErk1/2 and the isotype-matched control antibody (BD Biosciences).

Saito S., Kawamura T., Higuchi M., Kobayashi T., Yoshita-Takahashi M., Yamazaki M., Abe M., Sakimura K., Kanda Y., Kawamura H., Jiang S., Naito M., Yoshizaki T., Takahashi M, & Fujii M. (2015). RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells. European journal of immunology, 45(5).

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