Anti mhcii m5 114

Manufactured by Thermo Fisher Scientific

Anti-MHCII (M5/114.15.2) is a monoclonal antibody that binds to the major histocompatibility complex class II (MHC-II) molecules. This antibody can be used as a research tool to identify and study cells expressing MHC-II, which are typically found on the surface of antigen-presenting cells, such as B cells, dendritic cells, and macrophages.

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Lab products found in correlation

Anti cd3 17a2, by BioLegend (2 mentions) Anti cd11c n418, by Thermo Fisher Scientific (2 mentions) Anti siglecf e50 2440, by BD (2 mentions) Anti tyrosine hydroxylase, by Merck Group (1 mentions) Hardset mounting medium, by Vector Laboratories (1 mentions) Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Anti f4 80 bm8, by BioLegend (1 mentions) Anti foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions) Anti cd4 rm4 5, by BD (1 mentions) Anti gr 1 rb6 8c5, by BD (1 mentions) Anti cd44 im7, by BioLegend (1 mentions) Anti cd25 pc61, by BioLegend (1 mentions) Mel 14, by Thermo Fisher Scientific (1 mentions) Lsrii flow cytometer, by BD (1 mentions) 70 m cell strainer, by BD (1 mentions) Anti cd25 pc61, by Thermo Fisher Scientific (1 mentions) Cd16 32 fc block, by BD (1 mentions) Anti cd49b dx5, by Thermo Fisher Scientific (1 mentions) Anti cd19, by BioXCell (1 mentions) Facsaria 3, by BD (1 mentions)

Spelling variants of this product

MHCII (84 citations) Anti-MHCII (34 citations) M5/114.15.2 (19 citations) Anti-MHCII (clone M5/114) (2 citations) M5/114.15.2 (anti-MHCII, (2 citations)

6 protocols using anti mhcii m5 114

Quantification of Dopaminergic Neurons

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At 4 weeks and 6 months post-transduction, animals were deeply anesthetized and transcardially perfused, post-fixed for 24 hours, and cryoprotected as previously described (Harms, et al. 2013 (link)). Brains were frozen on dry ice and cryosectioned coronally on a sliding microtome (cut thickness: 40 μm); sections were collected serially throughout the striatum and SNpc, placed into tissue collection solution (50% 0.01 M PBS, 50% glycerol), and stored at −20°C for immunohistochemical analysis.
For fluorescent analysis, free-floating sections were labeled with anti-MHCII (M5/114.15.2, eBiosciences, 1:100) or anti-Tyrosine hydroxylase (TH) (Millipore, 1:2000) antibodies overnight at 4°C. Appropriate Alexa-conjugated secondary antibodies diluted 1:1000 (Life Technologies) were used at room temperature for 2.5 hours. Sections were mounted onto coated glass slides, and cover slips were added using Vectashield Hard Set mounting medium.
For TH neuron quantification using unbiased stereological analysis, free floating sections were stained as previously described (Harms, et al. 2013 (link); Luk, et al. 2012 (link); St Martin, et al. 2007 (link)), coded, and analyzed with an Olympus BX51 microscope and MicroBrighfield software (MicroBrightfield Inc., Williston, VT).

Harms A.S., Thome A.D., Yan Z., Schonhoff A.M., Williams G.P., Li X., Liu Y., Qin H., Benveniste E.N, & Standaert D.G. (2017). Peripheral Monocyte Entry is Required for Alpha-Synuclein Induced Inflammation and Neurodegeneration in a Model of Parkinson Disease. Experimental neurology, 300, 179-187.

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Multi-Dimensional Flow Cytometry Analysis

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Spleens were harvested and single-cell suspensions were prepared from each animal. Samples were stained with anti-CD3 (17A2, Biolegend, San Diego, CA), anti-CD4 (RM4.5, BD Bioscience), anti-CD8 (MCD0830, Invitrogen, Waltham, MA), anti-Gr-1 (RB6.8C5, BD Pharmingen, San Jose, CA), anti-B220 (RM2630, Invitrogen), anti-CD19 (CD5, Biolegend), anti-CD11b (M1-79, Biolegend), anti-CD11c-PE (HL3, eBioscience, Waltham, MA), anti-MHCII (M5/114.15.2, eBiosciences), anti-F4/80 (BM8, Biolegend), anti-CD25 (PC61, Biolegend), anti-FoxP3 (FJK-16S, eBioscience), anti-CD44 (IM7, Biolegend), and anti-CD62L (MEL-14, eBioscience). Similar techniques were used for flow cytometry on lung tissue. Samples were run on an LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software version10.0.7 (TreeStar, Ashland, OR).

Klingensmith N.J., Fay K.T., Lyons J.D., Chen C.W., Otani S., Liang Z., Chihade D.B., Burd E.M., Ford M.L, & Coopersmith C.M. (2019). Chronic alcohol ingestion worsens survival and alters gut epithelial apoptosis and CD8+ T cell function after Pseudomonas aeruginosa pneumonia-induced sepsis. Shock (Augusta, Ga.), 51(4), 453-463.

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Mouse Splenic T Cell Purification

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Spleens were dissected from mouse spleen using 70µm cell strainers (Falcon) and further processed under sterile conditions. Single-cell suspensions were rinsed-out with RPMI-1640 (Lonza) supplemented with 10% (vol/vol) fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 40 mM β-mercaptoethanol, 100 U ml−1of penicillin, and 100 U ml−1of streptomycin (all reagents from Lonza).
CD4⁺ cells were purified by negative selection with magnetic depletion of B cells, macrophages, DCs, NK cells, granulocytes and CD8⁺ cells using a cocktail of biotinylated antibodies (anti-CD49b, DX5, eBiosciences; anti-GR1, RB6-8C5, produced in house; anti-Ter119, BioXCell; anti-CD11c, N418, produced in house; anti-CD19, BioXCell; anti-CD8β, H35, produced in house; anti-CD25, PC61.5, eBiosciences (used for Tconv-but not Treg- purification); anti-MHCII, M5/114.15.2, eBiosciences). Cells were recuperated after flow-through the magnetic column, with previous incubation with anti-biotin Microbeads (Miltenyi Biotec). Untouched cells were stained to exclude dead cells and incubated with Fc receptor-blocking antibodies CD16/32 (Fc block; BD Pharmingen) and surface staining antibody CD3⁺ and CD4⁺. Tconvs and Tregs were identified in FSC/SSC-low-to-moderate and sorted as GFP^- or GFP⁺ respectively using a BD FACSAria III.

Bowakim-Anta N., Acolty V., Azouz A., Yagita H., Leo O., Goriely S., Oldenhove G, & Moser M. (2023). Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs. Frontiers in Immunology, 14, 1023064.

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Lung Cell Phenotyping in Chimeric Mice

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Preparation of lung cells for the determination of cell exchange in chimeric mice has been described [62 (link)]. The lung cell suspension was labelled with anti-panCD45 (30-F11, eBioscience), anti-CD45.1 (A20, BD Pharmingen), anti-CD45.2 (104, BD Pharmingen), anti-Ly6G (1A8, BD Pharmingen), anti-CD11c (N418, eBioscience) anti-MHC-II (M5/114.15.2, eBioscience), anti-Siglec-F, (E50-2440, BD Pharmingen) and anti-CD64 (X54-5/7.1, BD Pharmingen). Cells were analyzed on a Becton Dickinson LSRFortessa flow cytometer using FACSDIVA software (BD Biosciences).

Naujoks J., Tabeling C., Dill B.D., Hoffmann C., Brown A.S., Kunze M., Kempa S., Peter A., Mollenkopf H.J., Dorhoi A., Kershaw O., Gruber A.D., Sander L.E., Witzenrath M., Herold S., Nerlich A., Hocke A.C., van Driel I., Suttorp N., Bedoui S., Hilbi H., Trost M, & Opitz B. (2016). IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid. PLoS Pathogens, 12(2), e1005408.

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Comprehensive Immune Cell Profiling of BAL and Lung

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To analyze immune cell populations, BAL and lung cells were incubated with rat anti-mouse CD16/CD32 (BD Biosciences) blocking antibody for 10 min at RT. Then, the BAL cells were stained with anti-Gr-1 (RB6-8C5, BioLegend, San Diego, CA, United States), anti-Siglec-F (E50-2440, BD Biosciences), Ly6c (AL-21, BD Biosciences), anti-MHCII (M5/114.15.2, eBioscience, San Diego, CA, United States), anti-CD11b (M1-70, BioLegend), anti-CD14 (Sa14-2, BioLegend), anti-CD11c (N418, eBioscience), anti-CD49b (DX5, BioLegend), anti-PDCA-1 (eBio927, eBioscience), DAPI (BioLegend), and anti-CD45 (30-F11, BioLegend) antibodies for 30 min at 4°C in dark. The lung cells were also stained with anti-CD3 (17A2, BioLegend), anti-CD4 (RM4-5, BioLegend), anti-CD8 (53-6.7, BioLegend), and anti-CD45 (30-F11, BioLegend) antibodies for 30 min at 4°C in a dark place. The stained cells were fixed in FACS lysing solution (BD biosciences) and analyzed by BD LSR Fortessa (BD Biosciences). All flow cytometry data were analyzed by Flowjo software (TreeStar Inc., Ashland, OR, United States). The gating strategy for immune cell populations is described in Supplementary Figures S1, S2.

Lee Y.J., Lee J.Y., Jang Y.H., Seo S.U., Chang J, & Seong B.L. (2018). Non-specific Effect of Vaccines: Immediate Protection against Respiratory Syncytial Virus Infection by a Live Attenuated Influenza Vaccine. Frontiers in Microbiology, 9, 83.

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Isolation and Activation of CD4+ T Cells

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CD4 þ T cells from WT or l-MYC spleens were obtained using the EasySep Mouse CD4 þ T-Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer's instructions. Sorted CD4 þ cells were labeled with CPD, as described above, and stimulated (2 Â 10 5 /well) with irradiated (30 Gy) WT splenocytes (1.5 Â 10 5 /well) and rIL2 (50 U/mL) in the presence of irradiated (100 Gy) 291 or B16F0 cells (10 5 /well) in 96-well round-bottom plates. Alternatively, WT splenocytes were loaded with the indicated peptides (1 mg/mL; Metabion) for 2 hours at 37 C, washed and cocultured with sorted CD4 þ T cells. Where indicated, anti-MHC-II (M5/114.15.2; eBioscience) was included (diluted 1:1,000). After 7 days, cells were harvested, stained with anti-Foxp3 and anti-Ki-67 and analyzed by flow cytometry.

Ahmetlić F., Riedel T., Hömberg N., Bauer V., Trautwein N., Geishauser A., Sparwasser T., Stevanović S., Röcken M, & Mocikat R. (2019). Regulatory T Cells in an Endogenous Mouse Lymphoma Recognize Specific Antigen Peptides and Contribute to Immune Escape. Cancer immunology research, 7(4).

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