Krn7000

Manufactured by Avanti Polar Lipids

KRN7000 is a synthetic glycolipid compound. It serves as a potent activator of natural killer T cells (NKT cells) by binding to the CD1d protein. The compound is commonly used in research settings to study the role and function of NKT cells in various biological processes and disease models.

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Lab products found in correlation

Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Brdu flow kit, by BD (1 mentions) Ultrapure lps from e coli o111 b4, by InvivoGen (1 mentions) Fam flivo, by ImmunoChemistry Technologies (1 mentions) Anti mouse cd3 antibody, by BioLegend (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Bafilomycin a1, by Merck Group (1 mentions) C24 0, by Avanti Polar Lipids (1 mentions) C24 1, by Avanti Polar Lipids (1 mentions) Rat igg, by Merck Group (1 mentions) Sodium sulfite, by Merck Group (1 mentions) Tyloxapol, by Merck Group (1 mentions) Sulfatide, by Avanti Polar Lipids (1 mentions) αgalcer, by Avanti Polar Lipids (1 mentions)

4 protocols using krn7000

Evaluating iNKT Cell Activation and Apoptosis

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For BrdU labeling, mice were injected I.P. with 1 mg BrdU daily for six days. One day later, tissues were harvested and BrdU incorporation in iNKT cells was evaluated with the BrdU Flow Kit (BD Pharmingen) per the manufacturer’s instructions. As a cytokine-driven model of iNKT cell activation, mice were injected I.V. with 2mg ultra-pure LPS from E. coli O111:B4 (InvivoGen) per kg body weight. As an antigen-driven model of iNKT cell activation, mice were injected I.P. with 1 μg αGalCer analog KRN7000 (Avanti Polar Lipids). Both αGalCer and LPS were prepared in DMSO and diluted 1:10 (v/v) in sterile saline immediately prior to injection. Mice were sacrificed and tissues harvested after either 4 or 72 hours. In some experiments, mice were injected I.P. with 200μg ultra-low endotoxin azide-free anti-IFNγ (XMG1.2) or isotype control antibodies (Biolegend) 8 hours prior to αGalCer injection. For in vivo analysis of apoptosis mice were injected I.V. with the fluorescent poly-caspase binding reagent FAM-FLIVO (Immunochemistry Technologies) and analyzed per the manufacturer’s instructions. For ex-vivo intracellular cytokine staining, suspensions of SVF cells were cultured for two hours at 37°C in complete media in the presence of Brefeldin A prior to staining with flow cytometry antibodies.

LaMarche N.M., Kane H., Kohlgruber A.C., Dong H., Lynch L, & Brenner M.B. (2020). Distinct iNKT cell populations use IFNγ or ER stress-induced IL-10 to control adipose tissue homeostasis. Cell metabolism, 32(2), 243-258.e6.

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Synthetic Lipid Analogs for CD1d Activation

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Synthetic lipids (KRN7000, C24:0, C24:1, and βGalCer C24:1) were purchased from Avanti Polar Lipids (Alabaster). C24:2, pC24:0, pC24:1, pC24:2, and βGalCer C24:2 were synthesized in our laboratory at the University of Connecticut, as detailed in Supplemental Methods. All lipids were dissolved in the vehicle (0.5% polysorbate-20) and diluted in PBS or complete RPMI 1640 medium with 10% FBS. For the hybridoma assay, sulfatide analogs and βGalCer analogs were dissolved in DMSO (Life Technologies, Thermo Fisher Scientific). The purified anti-CD1D1 antibody (clone 20H2) was purchased from Harlan. Rat IgG was purchased from MilliporeSigma. The anti–mouse CD3 antibody was purchased from BioLegend (clone 145-2C11). Mouse CD1D1 monomers were provided by the NIH Tetramer Core Facility (Emory University, Atlanta, Georgia, USA). The fluorescent protein–labeled mAbs used for flow cytometry are detailed in the Supplemental Methods. Bafilomycin A1 (MilliporeSigma) was dissolved in DMSO and added to cell cultures at a final concentration of 50 nM, 5 minutes before adding lipid antigens. Sodium sulfite (MilliporeSigma) was dissolved in PBS and used at the concentrations shown.

Nishio K., Pasquet L., Camara K., DiSapio J., Hsu K.S., Kato S., Bloom A., Richardson S.K., Welsh J.A., Jiang T., Jones J.C., Cardell S., Watarai H., Terabe M., Olkhanud P.B., Howell A.R, & Berzofsky J.A. (2023). Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer. The Journal of Clinical Investigation, 134(4), e165281.

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Lipid Loading of CD1 Proteins

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Lipids were dissolved by sonication in TBS (10 mM Tris pH 8.0, 150 mM NaCl) containing 0.05 % (v/v) tyloxapol (Sigma) and stored at −20°C prior to use. Prior to loading, lipids were thawed and re-sonicated for 30 min. All loading was performed overnight at RT. For staining in figure 1, CD1d was loaded with α-GalCer (KRN7000) at a 3:1 molar ratio (lipid:CD1). For Figure 6, CD1d was loaded with PBS44 (provided by Paul Savage, Brigham Young University) at 6:1, LPC (C18:1) or sulfatide (C24:1), both at 12:1. CD1c was loaded with LPC (C18:1), sulfatide (C24:1), PC (C18:0-C18–1) or GD3 at 12:1. KRN7000, LPC, PC, sulfatide and GD3 were purchased from Avanti Polar Lipids. CD1b was loaded with GMM (Moody laboratory, Brigham and Women’s Hospital, Boston) at 6:1.

Gherardin N.A., Redmond S.J., McWilliam H.E., Almeida C.F., Gourley K.H., Seneviratna R., Li S., De Rose R., Nguyen-Robertson C.V., Su S., Ritchie M.E., Villadangos J.A., Moody D.B., Pellicci D.G., Uldrich A.P, & Godfrey D.I. (2021). CD36 family members are TCR-independent ligands for CD1 antigen-presenting molecules. Science immunology, 6(60), eabg4176.

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NKT Cell Activation by α-GalCer Analogues

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KRN7000 (alpha-galactosyl ceramide, α-GalCer) was purchased from Avanti Polar Lipids, Inc. Gal(α1–2)galactosylceramide (GalGalCer) and C20:2 (α-GalCer analogue with 20 carbon di-unsaturated N-acyl chain) were prepared from d-lyxose, as described (27 (link)). All lipids were diluted at a concentration of 1 mg/ml in dimethyl sulfoxide (DMSO). L cells were seeded in 96-well plates at a density of 4 × 10⁴ cells per well and lipids were added after 6 h. Cells were washed three times with 1× RPMI-1640 medium (Thermo Fisher Scientific) and 1x10⁵ NKT hybridoma DN32.D3 (28 ) cells per well added. After 24 h, the supernatant was obtained and IL-2 concentrations determined using a mouse interleukin (mIL2) enzyme-linked immunosorbent assay (ELISA) (BD Bioscience) according to the manufacturer’s instruction.

Rudolph M., Wang Y., Simolka T., Huc-Claustre E., Dai L., Grotenbreg G., Besra G.S., Shevchenko A., Shevchenko A, & Zeissig S. (2022). Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids. Frontiers in Immunology, 13, 897873.

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