Invitrogen

MHC Class I (H-2Kd/H-2Dd) Monoclonal Antibody (34-1-2S), eFluor™ 450, eBioscience™

12 Published Figures

View all (19) MHC Class I (H-2Kd/H-2Dd) antibodies

Questions & Answers

Questions & Answers

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

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MHC Class I (H-2Kd/H-2Dd) Antibody (48-5998-82) in Flow

Staining of BALB/c splenocytes with 0.5 µg of Mouse IgG2a K Isotype Control eFluor® 450 (Product # 48-4724-82) (blue histogram) or 0.5 µg of Anti-Mouse MHC Class I (H-2Kd/H-2Dd) eFluor® 450 (purple histogram). Cells in the lymphocyte gate were used for analysis.

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow)

Product Image Gallery

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Staining of BALB/c splenocytes with 0.5 µg of Mouse IgG2a K Isotype Control eFluor® 450 (Product # 48-4724-82) (blue histogram) or 0.5 µg of Anti-Mouse MHC Class I (H-2Kd/H-2Dd) eFluor® 450 (purple histogram). Cells in the lymphocyte gate were used for analysis.

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Fig. 3 IL11 activates STAT3 to inhibit IFNgamma-induced STAT1 signaling and MHC-I expression. A , B Immunoblot of STAT1/3 (total and phosphorylated) in IL11-treated (100/200 ng/ml) MC38 with/without IFNgamma induction (10 ng/ml). GAPDH as internal reference. STAT1 phosphorylation ratio was calculated. Data presented as means + SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, t test. C mRNA expression of CXCL9, H2-D1, H2-K1 in IL11-treated (100/200 ng/ml) MC38 with/without IFNgamma induction (10 ng/ml). Data presented as fold change to control group and means + SEM from three independent experiments. * P < 0.05, ** P < 0.01, t test. D , E Integrated graph and MFI (mean fluorescence intensity) of flow cytometry analysis on cell surface H2-Kb/Db of IL11-treated (100/200 ng/ml) MC38 with/without IFNgamma induction (10 ng/ml). Data presented as fold change to control group and means + SEM from three independent experiments. * P < 0.05, t test. F Representative images of murine intestinal organoids under IL11 stimulation. Scale bar, 400 mum. (G)-(H) Immunoblot of STAT1/3 (total and phosphorylated) in IL11-treated (100 ng/ml) murine intestinal organoids with/without IFNgamma induction (10 ng/ml). STAT1 phosphorylation ratio was calculated. Data presented as means + SD from three independent experiments. ** P < 0.01, *** P < 0.001, t test. I - K mRNA expression of CXCL9, H2-D1, H2-K1 in IL11-treated (100/200 ng/ml) Stat3 fl/fl organoids ( I ), HT-29 cell

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Fig. 1 SHP1 deficiency had no effect on the cell morphology, proliferation rate or surface markers. a Phase-contrast images of MSCs derived from bone marrow of WT and me v /me v mice. Scale bar: 50 mum. b The same number of WT and me v /me v MSCs were coated into the 6 well plates, the number were calculated every day until the sixth day and the proliferation rate was monitored. c After isolation from bone marrow and culture for an equal number of passages, me v /me v and WT MSCs were harvested and analyzed for the indicated markers by immunofluorescence staining and flow cytometry. The experiments were repeated at least three times. Error bars represent +- SEM (n = 3), ns, not significant

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Figure 7 Analysis of the contribution of JAK/STAT and NF-kB dependent signals on IFNgamma responsiveness in NSCLC lines. CMT167 (A-C) or LLC (D-F) cells were cultured for 48 hours with IFNgamma (100 ng/mL), in the presence or absence of various inhibitors. (A-C) CMT167 cells were cultured for 48 hours with IFN gamma (100 ng/mL), the JAK/STAT inhibitor ruxolitinib (1 uM), and/or the IkappaB kinase (IKK) inhibitor IKK-16 (500 nM). Cells were then analyzed by flow cytometry for surface protein expression as indicated. (D-F) LLC cells were cultured for 48 hours with IFNgamma (100 ng/mL), trametinib (10 nM), ruxolitinib (1 uM) and/or IKK-16 (500 nM). Cells were analyzed by flow cytometry for cell surface expression of (A, D) PD-L1, (B, E) MHC I and (C, F) MHC II. (G) LLC cells were cultured for 48 hours with interferon gamma (100 ng/mL) and/or trametinib at indicated concentrations. Cells were then collected, fixed, permeabilized and analyzed for intracellular total STAT1 levels. All flow cytometry data were gated on viable singlets, except G in which cells were fixed and gated on singlets. Two independent experiments were performed, with n=3 replicates total for each variable analyzed. Statistical analysis was done comparing the effect of inhibitors on induction by IFNgamma (A-C), by IFNgamma plus trametinib (D-F), or trametinib (panel G) using one-way ANOVA subjected to Sidak''s multiple comparisons test, with statistical significance indicated as follows: *p<0.05; **p<0.01; ***

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Figure 2 Interferon gamma responsiveness in mouse and human NSCLC cell lines. In A-C, cultured cell lines (CMT167, LLC, EA1, EA2) were treated with vehicle or 100 ng/mL IFNgamma for 48 hours, and analyzed by flow cytometry for cell surface expression of (A) PD-L1, (B) MHC I, or (C) MHC II. Data for PD-L1 and MHC I are defined by median fluorescent intensity (MFI) of all cells, given the uniform induction of these proteins ( online supplementary figure 1 ). MHC II expression is defined as the frequency of positive cells, based on MHC II induction in a subset of cells ( online supplementary figure 2 ). Statistical analysis in (A, B) defined between vehicle and IFNgamma treated samples using an unpaired T-test (statistical significance identified by *p<0.05). Statistical analysis in (C) compared the frequency of MHC II+events between cell lines with matched oncogene status (ie, KRAS mutant lines, CMT167 and LLC; EML4/ALK cell lines, EA1 and EA2). Statistical analysis was performed on (C) using a one-way ANOVA. For A-C, data are from four replicates performed over three independent experiments. For C, vehicle controls were uniformly <0.5% MHC II+for each of the cell lines and were not included on the figure. (D-H) Human NSCLC lines (H1299, H1650, H460) were treated with vehicle or 100 ng/mL IFNgamma for 48 hours, and analyzed by flow cytometry for cell surface expression of (D-E) PD-L1, (F-G) MHC I and (H) MHC II, quantifying either the MFI (D, F) or percentage of HLA-DR (ie, MHC

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Figure 1 High-dimensional mass cytometry analysis of murine NSCLC cells stimulated with IFNgamma. CMT167 and LLC cells were cultured under either basal or IFNgamma stimulated conditions (100 ng/mL) for 48 hours, harvested and stained with a panel of metal-conjugated antibodies. (A) PhenoGraph analysis of CMT167 (left) and LLC cells (right), in which dominant cell clusters present under basal or IFNgamma stimulation were identified. Cellular events were plotted on a tSNE plot, where each unique cluster is given a distinct color. (B) Analysis of protein expression in CMT167 (left) and LLC cells (right), with each plot depicting expression in cells cultured under basal (""basal"", outlined in thick black line on each plot) or IFNgamma stimulated conditions (''+IFNgamma'', outlined in thick red line on each plot). Each row depicts expression of a different protein, with scale on the right side depicting range from minimum (bottom) to maximum (top), calculated based on the equation arcsinh (x/f), where x is the signal intensity and f=5. Yellow asterisk in MHC II plot for CMT167 cells identifies a subpopulation of IFNgamma-treated cells that induce MHC II expression. PhenoGraph analysis was done on files that were normalized to equilibration beads, with all events defined as viable ( 195 Pt-), nucleated ( 191 Ir+ 193 Ir+) events. CMT167 and LLC cells, under basal and IFNgamma stimulated conditions were clustered together, using 10 000 events per sample and 27 markers. Data are from

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Figure 4 Trametinib enhances responsiveness to IFNgamma in CMT167 cells. CMT167 cells were treated with vehicle or IFNgamma, in the presence or absence of the MEK inhibitor, trametinib, at indicated concentrations for 48 hours. The cells were then collected, stained with the indicated antibody and analyzed using multiparameter flow cytometry for cell surface expression of (A, D) PD-L1 or (B, E) MHC I, defined by median fluorescent intensity (MFI) and (C, F, G) MHC II, defined by the frequency of MHC II positive events. (A-C) Depict studies where cells were either vehicle treated or stimulated with IFNgamma (100 ng/mL), with data in (D-G) further quantifying the effect of varying concentrations of either IFNgamma or trametinib on protein expression. (G) Representative flow cytometry dot plots showing the percentage of MHC II+events, defined relative to side scatter (SSC). All flow cytometry data were gated on viable, singlets with MFI as x10 6 values. Data are from three independent experiments. Statistical analysis was done comparing the effect of trametinib on IFNgamma induction using one-way ANOVA subjected to Sidak''s multiple comparisons test, with statistical significance indicated as follows: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Graphs show mean+-SEM. ANOVA, analysis of variance; IFNgamma, interferon gamma.

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Figure 6 Trichostatin A and trametinib cotreatment leads to increased expression of interferon responsive proteins in LLC cells. LLC cells were pretreated for 24 hours with vehicle, trametinib (10 nM) and/or trichostatin A (TSA, 50 nM). The following day, media was changed, and cells were treated with vehicle, trametinib, TSA, and/or IFNgamma for an additional 48 hours. Cells were then analyzed for cell surface expression of (A) PD-L1, (B) MHC I or (C-D) MHC II by flow cytometry. Data in panels A-D are from three independent experiments (mean+-SEM), with representative density plots in panel E showing effect of various treatments on MHC II expression. All flow cytometry data were gated on viable, singlets with MFI as x10 6 values. Statistical analysis was done comparing the effects of trametinib and TSA on IFNgamma induction using one-way ANOVA subjected to Sidak''s multiple comparisons test, with statistical significance indicated as follows: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; IFNgamma, interferon gamma; LLC, Lewis lung carcinoma; MFI, median fluorescent intensity; SSC, side scatter; TSA, trichostatin A.

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Figure 5 Trametinib enhances responsiveness to IFNgamma in LLC cells. LLC cells were treated with vehicle or IFNgamma (100 ng/mL), in the presence or absence of the MEK inhibitor, trametinib, at the indicated concentrations for 48 hours. Cells were then harvested and analyzed by flow cytometry for (A, B) PD-L1, (C, D) MHC I or (E, F) MHC II expression. Data depict median fluorescent intensity (MFI) with representative histograms to the right (B, D) or the frequency of MHC II+events with representative dot plots (E, F). Data are from three independent experiments. All flow cytometry data were gated on viable, singlets with MFI as x10 6 values. Statistical analysis was done comparing the effect of trametinib on IFNgamma induction using one-way ANOVA subjected to Sidak''s multiple comparisons test, with statistical significance indicated as follows: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Graphs show mean+-SEM. ANOVA, analysis of variance; IFNgamma, interferon gamma.

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Figure 1 Characterization of EGFP-AFSCs in culture. (A) Amniotic fluid stem cells (AFSCs) (third passage) exhibit fibroblast-like morphology and adhere to the surface of plastic cell culture dishes. (B) Harvested AFSCs express enhanced green fluorescence protein (EGFP). (C) Immunophenotypes of AFSCs at their third passage expressed markers of mesenchymal stem cells (MSCs) but not those of hematopoietic and endothelial cells. The black histograms indicate the respective isotype control. (D) Oil Red O staining after 14 days of culture in adipogenic medium showed adipogenic differentiation potential of AFSCs. (E) Alizarin Red S (ARS) staining after 21 days of culture in osteogenic medium showed osteogenic differentiation potential of AFSCs. (F) Toluidine blue O staining after 21 days culture in induction chrondrogenic medium showed chondrogenic differentiation potential of AFSCs. Scale bars = 100 um. (G) Proliferation ability of EGFP-AFSCs (third passage) was better than that of bone marrow MSCs (BMMSCs) (third passage) (n = 3). Data represent the mean +- standard error of mean (S.E.M.) * P <0.05. (H-J) Immunocytochemistry analysis revealed the OCT4 expression (red in I and J) by EGFP-AFSCs with nuclear counterstaining by 4',6-diamidino-2-phenylindole (DAPI) (blue in H, J and K). Scale bar = 25 um.

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Figure 5 Differentiation potential of EMSCs and BMMSCs. (A) Adipogenic differentiation of EMSCs and BMMSCs at the fifth passage. Adipogenic capability was characterized by Oil Red O staining after seven days induction. EMSCs showed larger and more lipid drops than BMMSCs. (B) Oil Red O staining of EMSCs and BMMSCs were quantified for comparison (n = 3). (C, D) Expression level of adipogenic related genes were assessed by qPCR. EMSCs showed higher adipogenic gene expression level after seven days induction (n = 3). (E) MHC I expression profile of EMSCs was analysed by flow cytometry after seven days adipogenic induction. After adipogenesis, EMSCs were still negative for MHC I expression. (F) Osteogenic differentiation of EMSCs and BMMSCs at the fifth passage. Osteogenic capability was characterized by ARS staining after seven days induction. EMSCs showed higher calcium deposition than BMMSCs. (G) Quantitative results of ARS were performed to compare the osteogenic capacity of EMSCs and BMMSCs (n = 3). (H, I) Expression level of osteogenic related genes were assessed by qPCR. EMSCs showed higher osteogenic gene expression level after seven days induction (n = 3). (J) MHC I expression profile of EMSCs was analysed by flow cytometry after seven days osteogenic induction. After osteogenesis, EMSCs were still negative for MHC I expression. (K) Chondrogenic differentiation of EMSCs and BMMSCs at the fifth passage. Chondrogenic capability was evaluated by histological section of micr

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Figure 3 Surface markers analysis of EMSCs after IFN-gamma treatment and during increased serial passages. (A) MHC I and MHC II expression profiles for EMSCs (passage five) were analysed under 10 ng or (B) 100 ng IFN-gamma treatment. EMSCs were positive for MHC I and MHC II after IFN-gamma treatment. (C) EMSCs surface antigen profiles were detected via flow cytometric analysis during increasing passages. The markers of CD73, CD166, SSEA-4 and Sca-1 were decreased with propagating. Respective isotype control is indicated by the dotted line.

MHC Class I (H-2Kd/H-2Dd) Antibody in Flow Cytometry (Flow) — Published figure using MHC Class I (H-2Kd/H-2Dd) monoclonal antibody (Product # 48-5998-82) in Flow Cytometry

Product Details

48-5998-82

Applications

Tested Dilution

Publications

Flow Cytometry (Flow)

1 µg/test

View 6 publications 6 publications

Product Specifications

Species Reactivity

Mouse

Published species

Not Applicable

Host/Isotype

Mouse / IgG2a, kappa

Recommended Isotype Control

Mouse IgG2a kappa Isotype Control (eBM2a), eFluor™ 450, eBioscience™

Class

Monoclonal

Type

Antibody

Clone

34-1-2S

Conjugate

eFluor™ 450 eFluor™ 450 eFluor™ 450

Excitation/Emission Max

405/445 nm View spectra

Form

Liquid

Concentration

0.2 mg/mL

Purification

Affinity chromatography

Storage buffer

PBS, pH 7.2

Contains

0.09% sodium azide

Storage conditions

4° C, store in dark, DO NOT FREEZE!

Shipping conditions

Ambient (domestic); Wet ice (international)

RRID

AB_2574093

Product Specific Information

Description: The 34-1-2S monoclonal antibody reacts with the mouse MHC class I, H-2Kd and H-2Dd. This cytotoxic antibody also cross-reacts with Kb, s, r, q, p.

Applications Reported: This 34-1-2S antibody has been reported for use in flow cytometric analysis.

Applications Tested: This 34-1-2S antibody has been tested by flow cytometric analysis of mouse splenocytes. This can be used at less than or equal to 1 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.

eFluor® 450 is an alternative to Pacific Blue®. eFluor® 450 emits at 445 nm and is excited with the Violet laser (405 nm). Please make sure that your instrument is capable of detecting this fluorochome.

Excitation: 405 nm; Emission: 445 nm; Laser: Violet Laser.

Filtration: 0.2 µm post-manufacturing filtered.

Target Information

This protein is an MHC class I alloantigen, which is involved in antigen presentation to T cells expressing CD3/TCR and CD8.

HLA and MHC antibodies play a significant role in Immunopeptidomics, facilitating the identification and characterization of neoantigens through high-performance liquid chromatography coupled to tandem Mass Spectrometry.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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Product References

Pharmacologic inhibition of IL11/STAT3 signaling increases MHC-I expression and T cell infiltration.

Journal of translational medicine

Xiong W,Chen Y,Zhang C,Li J,Huang H,Zhu Y,Deng G,Cheng J,Lin Y,Shi Z,Mou T

Published figure using MHC Class I (H-2Kd/H-2Dd) monoclonal antibody (Product # 48-5998-82) in Flow Cytometry

Mon Jun 26 00:00:00 EDT 2023

Cancer cell-intrinsic expression of MHC II in lung cancer cell lines is actively restricted by MEK/ERK signaling and epigenetic mechanisms.

Journal for immunotherapy of cancer

Neuwelt AJ,Kimball AK,Johnson AM,Arnold BW,Bullock BL,Kaspar RE,Kleczko EK,Kwak JW,Wu MH,Heasley LE,Doebele RC,Li HY,Nemenoff RA,Clambey ET

Published figure using MHC Class I (H-2Kd/H-2Dd) monoclonal antibody (Product # 48-5998-82) in Flow Cytometry

Wed Apr 01 00:00:00 EDT 2020

Phosphatase SHP1 impedes mesenchymal stromal cell immunosuppressive capacity modulated by JAK1/STAT3 and P38 signals.

Cell & bioscience

Jiang M,Ye J,Wang X,Li N,Wang Y,Shi Y

Published figure using MHC Class I (H-2Kd/H-2Dd) monoclonal antibody (Product # 48-5998-82) in Flow Cytometry

Thu Apr 14 00:00:00 EDT 2022

Amniotic fluid stem cells prevent follicle atresia and rescue fertility of mice with premature ovarian failure induced by chemotherapy.

PloS one

Xiao GY,Liu IH,Cheng CC,Chang CC,Lee YH,Cheng WT,Wu SC

Published figure using MHC Class I (H-2Kd/H-2Dd) monoclonal antibody (Product # 48-5998-82) in Flow Cytometry

Thu May 28 00:00:00 EDT 2015

Isolation and characterization of novel murine epiphysis derived mesenchymal stem cells.

PloS one

Cheng CC,Lian WS,Hsiao FS,Liu IH,Lin SP,Lee YH,Chang CC,Xiao GY,Huang HY,Cheng CF,Cheng WT,Wu SC

Published figure using MHC Class I (H-2Kd/H-2Dd) monoclonal antibody (Product # 48-5998-82) in Flow Cytometry

Fri Sep 07 00:00:00 EDT 2012

Dynamics of lung macrophage activation in response to helminth infection.

Journal of leukocyte biology

Siracusa MC,Reece JJ,Urban JF,Scott AL

Published figure using MHC Class I (H-2Kd/H-2Dd) monoclonal antibody (Product # 48-5998-82) in Flow Cytometry

Mon Dec 01 00:00:00 EST 2008

Bioinformatics

Protein Aliases: class I major histocompatibility antigen H-2Kd; H-2 class I histocompatibility antigen, D-B alpha chain; H-2 class I histocompatibility antigen, K-B alpha chain; H-2 class I histocompatibility antigen, K-D alpha chain; H-2 class I histocompatibility antigen, K-K alpha chain; H-2 class I histocompatibility antigen, K-Q alpha chain; H-2 class I histocompatibility antigen, K-W28 alpha chain; H-2D cell surface glycoprotein; H-2D(B); H-2K(B); H-2K(K); H-2K(Q); H2-DC1-beta transplantation antigen protein; H2DD; H2K1; H2KD; MHC class I antigen; MHC class I H-2Dp; MHC class I H2 antigen; MHC class I H2-K-b-alpha-2 cell surface glycoprotein; MHC class I heavy chain H2-K; MHC H2-D-q alpha-chain; MHC H2-K transplantation antigen; Q5k protein

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Gene Aliases: H-2D; H-2K; H-2K(d); H2-D; H2-D1; H2-K; H2-K1; K-f

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UniProt ID: (Mouse) P01899, (Mouse) P01901

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Entrez Gene ID: (Mouse) 14964, (Mouse) 14972

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receptor binding protein binding beta-2-microglobulin binding peptide binding peptide antigen binding T cell receptor binding poly(A) RNA binding TAP binding protein heterodimerization activity major histocompatibility complex protein

Process(es)

positive regulation of T cell mediated cytotoxicity immune system process antigen processing and presentation of peptide antigen via MHC class I antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-dependent immune response antigen processing and presentation antigen processing and presentation of exogenous peptide antigen via MHC class I defense response to bacterium inner ear development

Performance Guarantee

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