H 2kb

Manufactured by BioLegend

Sourced in United States

H-2Kb is a mouse major histocompatibility complex (MHC) class I molecule. It is responsible for presenting peptide fragments to cytotoxic T cells, which is a key step in the adaptive immune response.

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Lab products found in correlation

Facsaria 2, by BD (2 mentions) Ifn γ, by BioLegend (2 mentions) Cd62l, by BioLegend (2 mentions) H 2db, by BioLegend (2 mentions) Pd l1, by BioLegend (2 mentions) Lamp1, by Abcam (1 mentions) Fluorescently labeled secondary abs, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions) Lsm 700 inverted confocal microscope, by Zeiss (1 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions) Lsrii cytometer, by BD (1 mentions) Polyinosinic polycytidylic acid poly 1 c, by Merck Group (1 mentions) Anti mouse cd40 clone 1c10, by Southern Biotech (1 mentions) Uea 1, by Vector Laboratories (1 mentions) Magnetic bead conjugated anti cd45 antibody, by Miltenyi Biotec (1 mentions) Dnase 1, by Roche (1 mentions) Liberase, by Roche (1 mentions) Lsrfortessa x 20 cell analyzer, by BD (1 mentions) Cytofix cytoperm solution, by BD (1 mentions) Tnf α, by BioLegend (1 mentions)

Close spelling variants of this product

APC anti-mouse H-2Kb/H-2Db Anti-H-2Kb PE PE anti-mouse SIINFEKL-H-2Kb antibody Anti- H-2Kb bound to SIINFEKL-APC H-2Kb (clone 25-D1.16, H-2Kb PE H-2Kb (clone AF6-88.5) APC anti-mouse H-2Kb H-2Kb bound to SIINFEKL H-2Kb/H-2Db

11 protocols using h 2kb

Visualization of Immune Cell Interactions

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Primary hepatocytes were adhered onto 3T3 NIH fibroblast-coated glass coverslips and BMDCs were adhered onto poly-L-lysine-coated glass coverslips. At the end of the experimental procedures, cells were fixed in 4% paraformaldehyde solution, permeabilized in 3% BSA 0.1% saponin PBS and stained with primary Abs specific for MR-1 (AbD Serotec), EEA1 (BioConcept), LAMP-1 (Abcam), TAP1 (Santa Cruz Biotechnology), H-2Kb (BioLegend), or H-2Kb/SIINFEKL (eBioscience) followed by fluorescently labeled secondary Abs (Life Technologies) and fluorochrome-conjugated phalloidin (Life Technologies). Liver, spleen, lungs, and kidneys were harvested after perfusion of euthanized animals with HBSS (Life Technologies). Organs were fixed in 4% paraformaldehyde solution and frozen in OCT (Sakura). Ten micrometer thick sections were sliced and stained with primary Abs specific for PD-L1 or PD-L2 (eBioscience) and fluorescently labeled secondary Abs (Life Technologies) or left unstained. Samples were mounted using ProLong Gold antifade reagent with DAPI (Life Technologies), imaged with a LSM 700 inverted confocal microscope (Zeiss) and data were analyzed with ImageJ software. For flow cytometry, at the end of the experimental procedures, hepatocytes or BMDCs were washed in 2% FBS PBS and acquired on an LSR II cytometer (BD Biosciences) and data analyzed with FlowJo software (Tree Star).

Damo M., Wilson D.S., Watkins E.A, & Hubbell J.A. (2021). Soluble N-Acetylgalactosamine-Modified Antigens Enhance Hepatocyte-Dependent Antigen Cross-Presentation and Result in Antigen-Specific CD8+ T Cell Tolerance Development. Frontiers in Immunology, 12, 555095.

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Multiparameter Immune Cell Profiling

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Blood samples were obtained from the tail vein into heparin-PBS. After lysing red blood cells, cells were incubated with Fc-block followed by staining with the following antibodies purchased from BioLegend; H2K^b, H2K^d, CD45.1, CD45.2, TCRβ, CD4, CD8a, CD19, CD25, Gr1, and CD11b. Cells were acquired by LSRII (BD) and data was analyzed with FlowJo software (BD).

Jensen J.S., Bruun B, & Gahrn-Hansen B. (1999). Unexpected Cross-Reaction with Fusobacterium necrophorum in a PCR for Detection of Mycoplasmas. Journal of Clinical Microbiology, 37(3), 828-829.

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Flow Cytometry Immunophenotyping of Activated Murine Cells

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Rat anti-CD11b-PE (clone M1/70) was purchased from eBioscience (San Diego, CA). Anti-mouse H-2D^b, H-2K^b, and CD80 were purchased from BioLegend (San Diego, CA). Anti-mouse CD40 (clone 1C10) was purchased from Southern Biotech (Birmingham, AL). polyinosinic-polycytidylic acid (Poly(I:C)) and lipopolysaccharide (LPS) from Escherichia coli (E.coli) were purchased from Sigma Aldrich (St. Louis, MO).

Woodham A.W., Cheloha R.W., Ling J., Rashidian M., Kolifrath S.C., Mesyngier M., Duarte J.N., Bader J.M., Skeate J.G., Da Silva D.M., Kast W.M, & Ploegh H.L. (2018). Nanobody-antigen conjugates elicit HPV-specific antitumor immune responses. Cancer immunology research, 6(7), 870-880.

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Thymic Epithelial Cell Isolation Protocol

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For the analysis of TECs, minced thymuses were digested with 1 unit per ml Liberase (Roche) in the presence of 0.01% DNase I (Roche). Single-cell suspensions were stained for the expression of CD326 (EpCAM, BioLegend), CD45 (eBioscience), CD249 (Ly51, eBioscience), H-2K^b (BioLegend) and I-A^b (BioLegend), and for the reactivity with UEA-1 (Vector Laboratories). For the intracellular staining of β5t, surface-stained cells were fixed in 2% (g/vol) paraformaldehyde, permeabilized in 0.05% saponin, and stained with rabbit anti-β5t antibody followed by AlexaFluor488-conjugated anti-rabbit IgG antibody. For the isolation of TECs, CD45⁻ cells were enriched with magnetic bead conjugated anti-CD45 antibody (Miltenyi Biotec). For the analysis of thymocytes and splenocytes, cells were stained for the expression of CD4, CD8 and TCRβ (BioLegend). Multicolor flow cytometry and cell sorting were performed on FACSAriaII (BD Biosciences).

Uddin M.M., Ohigashi I., Motosugi R., Nakayama T., Sakata M., Hamazaki J., Nishito Y., Rode I., Tanaka K., Takemoto T., Murata S, & Takahama Y. (2017). Foxn1-β5t transcriptional axis controls CD8+ T-cell production in the thymus. Nature Communications, 8, 14419.

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Multiparameter Flow Cytometry Analysis

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Single cell suspensions of organs and tumors were stained with the fluorochrome-conjugated antibodies protein as described [21 –24 (link)]. For intracellular staining, the cells were first permeabilized with a BD Cytofix/Cytoperm solution for 20 minutes at 4 ℃. Direct or indirect staining of fluorochrome-conjugated antibodies included: CD4, CD8, CD44, CD62L, CD69, Foxp3, and H-2K^b, IFNγ, TNFα, and IL-17A (BioLegend, or BD Biosciences, San Jose, CA, San Diego, CA). The samples were analyzed on a FACSCalibur or LSRFortessa X-20 Cell Analyzer (BD Biosciences). Data analysis was done using FlowJo software (Ashland, OR).

Cui C., Tian X., Lin Y., Su M., Chen Q., Wang S.Y, & Lai L. (2018). In vivo administration of recombinant BTNL2-Fc fusion protein ameliorates graft-versus-host disease in mice. Cellular immunology, 335, 22-29.

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Immunophenotyping Mouse Lymphocytes

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Antibodies for flow cytometry were obtained from BD Biosciences (CD8α, CD4, B220, TCRβ, CD44, NK1.1, Qa-1^b, CD11c, H2-K^b, H2-D^b, I-A^b and IFNγ ) and BioLegend (CD62L). All peptides were synthesized by Dr. David King (UC Berkeley).

Nagarajan N.A., de Verteuil D.A., Sriranganadane D., Yahyaoui W., Thibault P., Perreault C, & Shastri N. (2016). ERAAP shapes the peptidome associated with classical and non-classical MHC class I molecules. Journal of immunology (Baltimore, Md. : 1950), 197(4), 1035-1043.

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Multiparameter Flow Cytometry Analysis

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Cells were stained with combinations of fluorophore-conjugated antibodies against CD11b, CD11c, CD69, CD8, H-2K^b, H-2K^b-SIINFEKL complex (Biolegend), Gr-1 and IFN-γ (BD). For intracellular staining of IFN-γ, cells were incubated for 4 h at the presence of Leukocyte Activation Cocktail (BD), fixed with 4% paraformaldehyde and permeabilized with BD Perm/Wash buffer, followed by IFN-γ antibody staining. For H-2K^b-SIINFEKL Tetramer staining, cells were incubated at 4°C for 1 h. Alexa 488-labeled H-2K^b - SIINFEKL Tetramer was kindly provided by the NIH Tetramer Core at Emory University (Atlanta, GA, USA). Data were collected with a CyAn flow cytometer (Dako) or an Accuri C6 flow cytometer (BD) and analyzed with FlowJo software (Tree Star). Gating strategies were shown in Supporting Information Fig. 8.

Zhang W., Zhang C., Li W., Deng J., Herrmann A., Priceman S.J., Liang W., Shen S., Pal S.K., Hoon D.S, & Yu H. (2014). CD8+ T-cell immunosurveillance constrains lymphoid pre-metastatic myeloid cell accumulation. European journal of immunology, 45(1), 71-81.

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Dendritic Cell-Mediated Tumor Cell Killing

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MutuDC1s and MutuDC2s were exposed to 1 μg/mL LPS, 10 μg/mL IFNα, 5 μg/mL PolyI:C, or 0.5 μM CpG for 12 h, then rinsed and co-cultured with SYTO62-labeled B16 cells for 45 min. The SYTO62 signal in control and treated MutuDC1 and MutuDC2 was determined by flow cytometry. The activation of the DCs was confirmed based on morphological changes by microscopy and by flow cytometry with the following markers: CD40, CD80, CD86, and H2-K^b (BioLegend). In separate experiments, the B16 cells were left untreated or exposed to 1 μg/mL LPS or 10 μg/mL IFNα for 12 h, then labeled with SYTO62 dye and co-cultured with unmanipulated MutuDC1s.

Herbst C.H., Bouteau A., Menykő E.J., Qin Z., Gyenge E., Su Q., Cooper V., Mabbott N.A, & Igyártó B.Z. (2024). Dendritic cells overcome Cre/Lox induced gene deficiency by siphoning cytosolic material from surrounding cells. iScience, 27(3), 109119.

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Single Cell Immunophenotyping of MOC2 Tumors

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Only fresh cultured cells or tissue prepared into single cell suspensions were analyzed. The harvest and digestion of MOC2 tumors into single cell suspensions for analysis was performed as previously described [18 ]. Nonspecific surface staining was minimized by staining with CD16/32 (FcR) blocking antibodies for 10 min prior to the addition of primary antibodies when applicable. Anti-mouse NK1.1, CD3, CD16, NKG2D, CD44, CD27, PD-L1, PD-1, Tim-3, CTLA-4, H-2K^b, H-2D^b, pan-RAE, H60 and MULT-1 antibodies were from Biolegend. For some experiments, pre-treatment of cells with interferon γ (IFNγ) (20 ng/mL × 24 h) or YAC cells were used as positive controls. Primary antibodies were applied for 30–60 min at concentrations titrated for each antibody. Dead cells were excluded via 7AAD uptake and a “fluorescence-minus-one” technique was used to validate specific staining in all antibody combinations. Intracellular staining was performed with the eBioscience Intracellular Fixation and Permeabilization Buffer Set per manufacturer protocol. Granzyme B and IFNγ antibodies were from Biolegend. All analyses were performed on a BD FACSCanto analyzer running FACSDiva software and interpreted using FlowJo (vX10.0.7r2).

Friedman J., Morisada M., Sun L., Moore E.C., Padget M., Hodge J.W., Schlom J., Gameiro S.R, & Allen C.T. (2018). Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies. Journal for Immunotherapy of Cancer, 6, 59.

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CRISPR-Mediated Knockout of Cd274 in LN6-987AL Cells

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Cd274 was knocked out of the LN6-987AL cell line
using the lentiCRISPR v2 plasmid (Sanjana et
al., 2014). lentiCRISPR v2 was a gift from Feng Zhang (Addgene
plasmid # 52961). Lentiviral packaging and transduction were performed as
described above. All other genes were knocked out by transient transfection
with PX458 (Ran et al., 2013 (link))
followed by FACS sorting for GFP. pSpCas9(BB)-2A-GFP (PX458) was a gift from
Feng Zhang (Addgene plasmid # 48138). Following expansion, edited cells were
stained for PD-L1 (BioLegend, 10F.9G2) or H-2K^b (BioLegend,
AF6-88.5) and H-2D^b (eBioscience, 28-14-8) and subjected to two
rounds of sorting on a FACSAria II (BD). Guide RNAs were designed to
minimize off-target effects following previously described approaches (Doench et al., 2016 ; Hsu et al., 2013 ). Guide RNA sequences are listed
in Table S4.

Reticker-Flynn N.E., Zhang W., Belk J.A., Basto P.A., Escalante N.K., Pilarowski G.O., Bejnood A., Martins M.M., Kenkel J.A., Linde I.L., Bagchi S., Yuan R., Chang S., Spitzer M.H., Carmi Y., Cheng J., Tolentino L.L., Choi O., Wu N., Kong C., Gentles A.J., Sunwoo J.B., Satpathy A.T., Plevritis S.K, & Engleman E.G. (2022). Lymph node colonization induces tumor-immune tolerance to promote distant metastasis. Cell, 185(11), 1924-1942.e23.

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