Mhcii apc

Manufactured by Thermo Fisher Scientific

Sourced in United States

MHCII-APC is a laboratory instrument designed for the detection and analysis of major histocompatibility complex class II (MHCII) proteins. It utilizes allophycocyanin (APC) as a fluorescent label to enable the visualization and quantification of MHCII expression on cell surfaces.

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Lab products found in correlation

Cd86 pe, by Thermo Fisher Scientific (2 mentions) Cd44 pe, by Thermo Fisher Scientific (2 mentions) Cd80 fitc, by Thermo Fisher Scientific (2 mentions) Cd19 pe cy7, by BD (2 mentions) Facscanto 2, by BD (2 mentions) Tlr3 agonist poly 1 c, by InvivoGen (1 mentions) Abca1, by Novus Biologicals (1 mentions) Anti mouse hrp, by Santa Cruz Biotechnology (1 mentions) Anti rabbit hrp, by Santa Cruz Biotechnology (1 mentions) Cd11b percp cy5, by BioLegend (1 mentions) Gapdh, by Abcam (1 mentions) H3k27ac, by Abcam (1 mentions) Fixation and permeabilization buffer, by Thermo Fisher Scientific (1 mentions) Rabbit anti mouse relmα, by Thermo Fisher Scientific (1 mentions) Rat igg, by Merck Group (1 mentions) Cxcl1 kc, by R&D Systems (1 mentions) F4 80 percp cy5, by BioLegend (1 mentions) Ly6c apc cy7, by BioLegend (1 mentions) Cxcl2 mip 2, by R&D Systems (1 mentions) Softmax pro 6, by Molecular Devices (1 mentions)

11 protocols using mhcii apc

Macrophage Immunological Profiling

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The following antibodies and conjugates were used in this study: anti‐F4/80 clone Cl:A3‐1. LXRα/β antiserum,^[⁴³ (link)
^] LXRα (Abcam#PPZ0412), H3K27Ac (Abcam#ab4729), NOS‐2 (Santa Cruz#SC‐650 M‐19), ABCA1 (Novus Biologicals #NB400‐105), GAPDH (Abcam #ab9485‐100), Anti‐mouse‐HRP (Santa Cruz#SC‐2005), Anti‐rabbit‐HRP (Santa Cruz#SC‐2004), CD11b‐PerCP‐Cy5.5 (Biolegend #clone M1/70), F4/80‐PE or FITC (Ebioscience #clone BM8), Ly6G‐PE or FITC (BD Pharmingen #clone AL21), MHC‐II‐APC (eBioscience #M5/114.15.2). The following pharmacological reagents were obtained from the MRC PPU Reagents University of Dundee, UK: BI605906 (IKKβ inhibitor), 5Z‐7‐oxozeanol (TAK1 inhibitor), MRT67307 (TBK1 inhibitor) and the following products from Calbiochem: PD0325901 (MEK/ERK inhibitor), PD98059 (MEK1 and MEK2 inhibitor), PIK‐75‐hydrochloride (inhibitor of p110α subunit of PI3‐Kinase), SB590885 (Raf‐1 inhibitor); HX531 (RXR inhibitor). TLR agonists, Poly I:C (TLR3 agonist) and ultrapure LPS from E. coli 0111:B4 strain‐ (TLR4 ligand) were obtained from Invivogen and were used at 10 µg ml⁻¹ and 100 ng ml⁻¹ respectively.

Vladimir de la Rosa J., Tabraue C., Huang Z., Orizaola M.C., Martin‐Rodríguez P., Steffensen K.R., Zapata J.M., Boscá L., Tontonoz P., Alemany S., Treuter E, & Castrillo A. (2024). Reprogramming of the LXRα Transcriptome Sustains Macrophage Secondary Inflammatory Responses. Advanced Science, 11(20), 2307201.

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Flow Cytometric Analysis of Immune Cells

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For flow cytometric analysis, cells were blocked with PBS containing 1% bovine serum albumin and 0.1% rat IgG (Sigma-Aldrich, St. Louis, USA) for 30 min. After a washing step, cells were stained with SiglecF-PE or -AlexaFluor647 (Clone: E50-2440, BD Pharmingen, San Diego, USA), F4/80-PerCP-Cy5.5 (Clone: BM8), Ly6G-PE (Clone: 1A8), Ly6C-APC-Cy7 (Clone HK1.4) (all BioLegend, San Diego, USA), and CD11b-FITC or -PE-Cy7 (Clone: M1/7; eBioscience, San Diego, USA). For intracellular staining, cells were incubated with fixation and permeabilization buffer (eBioscience) overnight. Cells were stained with rabbit anti-mouse RELMα (Peprotech, Rocky Hill, USA) followed by donkey anti-rabbit IgG AlexaFluor647 (Clone: Poly4064, BioLegend) and CD86-PE (Clone: GL1) and MHCII-APC (Clone: M5/114.15.2, eBioscience) to determine cell activation. The gating strategy used to identify macrophages, monocytes, eosinophils and neutrophils is shown in Figure 1 (Fig. 1).
IFNγ , IL-10, TGFβ and TNF (all eBioscience) as well as CXCL1/KC and CXCL2/MIP-2 (both R&D, Minneapolis, USA) were measured from peritoneal lavage and serum by ELISA according to the manufacturers’ protocols and analyzed using a plate reader (Molecular Devices) with SoftMax Pro 6.

Buerfent B.C., Ajendra J., Stamminger W., Gondorf F., Hoerauf A, & Hübner M.P. (2019). TGFβ depletion does neither modulate acute E. coli-induced inflammatory immune responses nor impair the protective effect by chronic filarial infection. GMS Infectious Diseases, 7, Doc04.

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Lung Cell Isolation and Immunophenotyping

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Single-cell suspensions of the lung tissues were prepared by cutting them into small pieces followed by incubation in Dulbecco’s Modified Eagle Media (DMEM) containing 0.18 mg/mL Collagenase Type I (Sigma, St. Louis, MO, USA), 0.02 mg/mL DNase I (Sigma, St. Louis, MO, USA) for 1 h at 37 °C under constant rotation, followed by being mechanically passed through a 100 μm and 70 μm cell strainer sequentially. Erythrocytes were lysed using RBC lysis buffer (155 mM NH₄Cl, 12 mM NaHCO₃, 0.1 mM EDTA). Cells were then counted and subjected to flow cytometry. Lymphoid and myeloid compartments were investigated in the lung samples of mice on various intervention diets. Antibodies used for flow cytometry analysis were as follows: CD64-PeCy7 (Clone X54-5/7.1), Ly6C-PerCPCy5.5 (Clone AL-21), CD11b-V450 (Clone M1/70), MHCII-APC (Clone M5/114.15.2), CD103-PE (Clone M290), CD11c-A700 (Clone HL3), SiglecF-APCCy7 (Clone E5-2440), Ly6G-FITC (Clone 1A8), PD-1-FITC (Clone 29F.1A12), CD4-BV510 (Clone RM4-5), CD44-PE (Clone IM7), NK1.1-APCCy7 (Clone PK136), CD3-A700 (Clone 500A2), CD62L-V450 (Clone MEL-14), CD19-PerCPCy5.5 (Clone 1D3), CD8-APC (Clone 53-6.7), and KLRG1-BV786 (Clone 2F1) purchased from BD (Biosciences, Johannesburg, SA) and eBioscience (ThermoFisher, Johannesburg, SA) [29 (link),30 (link)].

Nienaber A., Baumgartner J., Dolman R.C., Ozturk M., Zandberg L., Hayford F.E., Brombacher F., Blaauw R., Parihar S.P., Smuts C.M, & Malan L. (2020). Omega-3 Fatty Acid and Iron Supplementation Alone, but Not in Combination, Lower Inflammation and Anemia of Infection in Mycobacterium tuberculosis-Infected Mice. Nutrients, 12(9), 2897.

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Dendritic Cell Phenotypic Analysis

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The single-cell suspension of DCs was collected from different treatments and referred to BMDCs stimulation test. For the staining of DCs, CD40-Pcy5.5E (0.01 μg/μL), CD86-PE (0.00125 μg/μL), MHC II-APC (0.0025 μg/μL), CD80-FITC (0.0025 μg/μL) (all from eBioscience) were used by the manufacturer's protocol (San Diego, CA). After staining, flow cytometric analysis was performed on the BD FACSVerse (BD Biosciences, San Jose, CA) flow cytometer and data were analyzed by Flowjo software (Ishizuka et al., 2019 (link); Jaaks et al., 2022 (link)).

Wu Y., Li Y., Zhao J., Wu Y., Lu D., Jia J., Chen T., He M., Lin J, & Yang Q. (2023). IBV QX affects the antigen presentation function of BMDCs through nonstructural protein16. Poultry Science, 102(5), 102620.

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Multiparametric Flow Cytometry Analysis

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Erythrocytes-depleted spleen cells, MLN cells and PECs were analyzed using a 6-color FACS, following a standard protocol (34 (link)). Cells were stained with different combinations of conjugated monoclonal antibodies CD3-FITC (Biolegend, San Diego, CA, USA), CD19-PEcy7, CD8-APCcy7, Sca-1-PE, CD25-APC, CD69-PEcy7, CD86-PE (BD Biosciences), CD11b-APCcy7, CD11c-APC, CD4-PE, F4/80-PEcy7, MHC II-APC (eBioscience, San Diego, CA, USA), and CD40-FITC (Southern Biotech). In the first panel, cells were immunphenotyped using mAbs to CD3, CD19, CD11b, CD11c, and Sca-1. A second panel was used to analyze T cell subsets and their activation status and consisted of mAbs to CD3, CD4, CD8, CD25, and CD69. In the third panel, myeloid cell activation status was analyzed using mAbs to CD11b, F4/80, CD40, CD86, and MHC class II. Non-viable cells that stained positive for 7-AAD (eBioscience), were excluded from the analysis. For each antibody, appropriate isotype control was used. Data were collected on 30,000 cells using BD FACS Canto II (BD Biosciences) and analyzed using BD FACSDiva software (BD).

Al-Barazie R.M., Bashir G.H., Qureshi M.M., Mohamed Y.A., Al-Sbiei A., Tariq S., Lammers W.J., al-Ramadi B.K, & Fernandez-Cabezudo M.J. (2018). Cholinergic Activation Enhances Resistance to Oral Salmonella Infection by Modulating Innate Immune Defense Mechanisms at the Intestinal Barrier. Frontiers in Immunology, 9, 551.

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Multicolor Flow Cytometry Analysis of Immune Cells

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Dimethyl sulfoxide (D8418), Hoechst 33342 (B2261), Tween 80 (P8074), and paraformaldehyde (158127) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, penicillin, streptomycin, and fetal bovine serum (FBS) were products of ThermoFisher/Gibco (Carlsbad, CA, USA). CPT-11 (#HY-16562A) was purchased from MedChem Express (Princeton, NJ, USA), dissolved in ethanol before freshly diluted in phosphate-buffered saline (PBS) containing 2% Tween 80 in each experiment, and the final concentration of ethanol, never exceeded 5%. Fluorescence-labeled monoclonal antibodies CD11b-FITC (#11-0112), F4/80-PE (#12-4801), MHCII-APC (#17-5321), CD23-PE (#12-0232), CD19-eFluor660 (#50-0193), and CD11b-AlexaFluor488 (#53-0112) were obtained from eBioscience (San Diego, CA, USA); monoclonal anti-mouse F4/80-AlexaFluor 647 (#123121) were bought from BioLegend (San Diego, CA, USA). The rabbit monoclonal antibody against GATA6 (#5851) was purchased from Cell Signaling Technology (Danvers, MA, USA). CF568 goat-anti-rabbit IgG (H+L), highly cross-adsorbed (#201031) was obtained from Biotium (Hayward, CA, USA).

Bai W.J., Li C.G., Zhang C.C., Xu L.H., Zeng Q.Z., Hu B., Hong Z., He X.H, & Ouyang D.Y. (2018). Prolonged Deleterious Influences of Chemotherapeutic Agent CPT-11 on Resident Peritoneal Macrophages and B1 Cells. Frontiers in Immunology, 8, 1919.

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Generating Bone Marrow-Derived Dendritic Cells

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BMDC were generated from bone marrow progenitor cells as described previously^{43 (link)}. Briefly, progenitor cells were obtained by flushing tibia and femur from BALB/c mice and cultured at 37 °C under 5% CO₂ in Iscove’s modified Dulbecco’s media supplemented by FCS (10%), gentamycin (50 µg/ml), glutamine (2 mM), β-mercaptoethanol (50 µM) and 10% of supernatant from a granulocyte–macrophage colony-stimulating factor (GM-CSF)-expressing J588 myeloma cell line for 10 days. On day 10, cells were stimulated by bacteria (ratio bacteria/cell: 10:1). After 24 h stimulation, BMDC were harvested, washed and stained using mAbs anti-CD11c PE-Cy7, CD40 PE, CD80 FITC, CD86 PE-Cy5, MHCII APC (eBioscience, San Diego, CA, USA) and acquired on BD FACS Canto II (Becton Dickinson).

Hrdý J., Couturier-Maillard A., Boutillier D., Lapadatescu C., Blanc P., Procházka J., Pot B., Ryffel B., Grangette C, & Chamaillard M. (2022). Oral supplementation with selected Lactobacillus acidophilus triggers IL-17-dependent innate defense response, activation of innate lymphoid cells type 3 and improves colitis. Scientific Reports, 12, 17591.

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Multiparameter Flow Cytometry Profiling

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For flow cytometry, we used monoclonal antibodies CD11c-FITC, CD80-PE, MHC II-APC, CD4-PE-Cyanine7, CD44-PE, CD45-FITC, Foxp3-PE, CD25-APC, and IL-17A-APC (eBioscience, USA). Cells were incubated with monoclonal antibodies for 30 min in the dark for the staining of surface antigens. IL-17A and Foxp3 were performed by intracellular staining.

Yuan D.H., Jia Y., Hassan O.M., Xu L.Y, & Wu X.C. (2020). LPS-Treated Podocytes Polarize Naive CD4+ T Cells into Th17 and Treg Cells. BioMed Research International, 2020, 8587923.

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Cabozantinib Modulates Immune Populations

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To analyze immune cell populations in the presence or absence of cabozantinib, CEA-Tg C57BL/6 mice (n =5/group) were fed control or cabozantinib-containing diet for 10 or 35 days. Spleens were harvested and adjusted to a single-cell suspension. Red blood cells were removed using ACK Lysing Buffer (Quality Biologicals Inc., Gaithersburg, MD). Remaining splenocytes were blocked with mouse Fc Block (BD Biosciences) for 30 min at 4°C, then stained with the following antibodies: CD3e-V500, CD4-AF700, CD8a-Pacific Blue, CD25-FITC, CD11b-V500, Gr-1-AF700, CD49b-FITC, CD19-PE-Cy7, CD11c-PerCP-Cy5.5 (BD Biosciences) and MHC II-APC (eBioscience) for 60 min at 4°C. For intracellular staining of cells with FoxP3-PE, cells were incubated with Fixation/Permeabilization solution for 16 h at 4°C, then incubated with the antibody in Permeabilization Buffer (eBioscience) for 60 min at room temperature. All samples were acquired on an LSR II flow cytometer and analyzed using FlowJo software.

Kwilas A.R., Ardiani A., Donahue R.N., Aftab D.T, & Hodge J.W. (2014). Dual effects of a targeted small-molecule inhibitor (cabozantinib) on immune-mediated killing of tumor cells and immune tumor microenvironment permissiveness when combined with a cancer vaccine. Journal of Translational Medicine, 12, 294.

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Characterization of Immune Cell Subsets

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H₂O₂ and CT were obtained from Sigma-Aldrich (St. Louis, MI, USA). Anti-mouse CD3-APC, CD4-FITC, CD8-PE, CD11c-eFluor 450, MHCII-APC, CD5-FITC and B220-PerCP-eFluor™ 710 antibodies were bought from eBioscience (San Diego, CA, USA). Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgA, HRP-conjugated goat anti-mouse IgG, HRP-conjugated goat anti-mouse IgG1 and HRP-conjugated goat anti-mouse IgG2a were obtained from Southern Biotech (Birmingham, AL, USA). RNA-easy Isolation was purchased from Vazyme Biotech Co., Ltd. (Nanjing, Jiangsu Province, China). SYBR qPCR Master Mix for quantitative real-time PCR (qRT-PCR) was bought from Yeasen Biotechnology Co., Ltd. (Shanghai, China). TMB was purchased from Beyotime Biotechnology (Shanghai, China). Red blood cell lysis buffer was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Silane coupling agent (KH550) and ZnONPs (purity 99.9%) were the products of Sinopharm Chemical Reagent Ltd. (Shanghai, China). The purified AMP was prepared in our laboratory (Yangzhou, Jiangsu Province, China). H₂SO₄, alcohol and HCl were obtained from Hushi Laboratory Equipment Co., Ltd. (Shanghai, China). Elisa plate was bought from cellpro biotechnology Co., Ltd. (Suzhou, Jiangsu Province, China).

Liu X., Lin X., Hong H., Wang J., Tao Y., Huai Y., Pang H., Liu M., Li J, & Bo R. (2024). Polysaccharide from Atractylodes macrocephala Koidz Binding with Zinc Oxide Nanoparticles as a Novel Mucosal Immune Adjuvant for H9N2 Inactivated Vaccine. International Journal of Molecular Sciences, 25(4), 2132.

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