Mhcii pe cy7

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The MHCII-PE-Cy7 is a fluorophore-conjugated antibody that specifically binds to the major histocompatibility complex class II (MHC II) molecules. It is designed for flow cytometry applications to identify and analyze cells expressing MHC II on their surface.

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7 protocols using mhcii pe cy7

Immunophenotyping of Activated BMDCs and Macrophages

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BMDC and MØ incubated with or without the peptides (5, 10 or 20 µg/mL) were washed with FACS buffer twice. Then, supernatants were removed and FACS buffer with Live/Dead – BV450 (BD Horizon); MHC-II – PE-Cy7 (clone: M5/114.15.2; Biolegend); F4/80 – FITC (clone: BM8; Biolegend); CD11c – BV711 (clone: N418; Biolegend); CD80 – PE (clone: 16-10A1; eBioscience); CD86 – APC (clone: GK1.5; Biolegend) were added for immunophenotyping and assessment of activation. The cells were incubated for 30 min at 4°C in the dark, washed 2X with FACS buffer and fixed with 1% paraformaldehyde. Sample acquisition was performed on a flow cytometer FACS BD LSRII housed in the Flow Cytometry Core Facility at the Einstein.

Silva L.B., Taira C.L., Cleare L.G., Martins M., Junqueira M., Nosanchuk J.D, & Taborda C.P. (2021). Identification of Potentially Therapeutic Immunogenic Peptides From Paracoccidioides lutzii Species. Frontiers in Immunology, 12, 670992.

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Multicolor Flow Cytometry Analysis

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The antibodies used for all experiments included CD11b-APCCy7 (#101226), Ly6G-PECy7 (#127618), CD3-APCCy7 (#100330), F4/80-APC (#123116), CD206-PE (#141706), MHCII-PE (#107647), MHCII-PECy7 (#107629), CD11c-APC (#117309), CD86-PECy7 (#105014) from Biolegend (Biolegend, San Diego, California, USA) and Ly6C-APC (#560595) from BD (BD Biosciences, UK). BD FACSDiva Software (Becton Dickinson) was used to acquire data. Analysis of data involved postacquisition gating using FlowJo software (Tree Star: Ashland, Oregon, USA).

Fan R., Satilmis H., Vandewalle N., Verheye E., Vlummens P., Maes A., Muylaert C., De Bruyne E., Menu E., Evans H., Chantry A., De Beule N., Hose D., Törngren M., Eriksson H., Vanderkerken K., Maes K., Breckpot K, & De Veirman K. (2023). Tasquinimod suppresses tumor cell growth and bone resorption by targeting immunosuppressive myeloid cells and inhibiting c-MYC expression in multiple myeloma. Journal for Immunotherapy of Cancer, 11(1), e005319.

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Multiparametric Flow Cytometry of Particle Uptake

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Cells collected from BALF and single cell suspensions collected from homogenized whole lung were stained with a panel of fluorescent antibodies for flow cytometric analysis of particle uptake and cell populations. The following antibodies were used for staining (BioLegend): CD45-PacBlue, CD11c-PE, MHCII-PE-Cy7, and Ly6-G-AF700. Cells were fixed with 4% PFA in PBS after staining and kept at 4°C until analysis. Flow cytometry data was collected on the LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
To correct for slight differences in fluorescence between particle types, relative particle fluorescence was independently determined by using a SpectraMax M5 plate-reader. Particles containing DyLight650 were incubated at 50 μg/mL in PBS and fluorescent intensity of each particle size was measured at 672 nm following excitation at 652 nm. Fluorescence values from all particle sizes were then normalized to that of the 80×320 particles and this ratio was used to adjust median fluorescence intensity (MFI) flow cytometry results.

Shen T.W., Fromen C.A., Kai M.P., Luft J.C., Rahhal T.B., Robbins G.R, & DeSimone J.M. (2015). Distribution and Cellular Uptake of PEGylated Polymeric Particles in the Lung Towards Cell-Specific Targeted Delivery. Pharmaceutical research, 32(10), 3248-3260.

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Cardiac immune cell isolation and characterization

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Hearts were thoroughly perfused with PBS, minced into 1 mm³ fragments and digested with 1 mg ml⁻¹ collagenase II (Worthington) in DMEM. Fragments were submitted to 5–6 cycles of digestion under gentle agitation at 37 °C. The supernatant was collected after every cycle and stored in DMEM with 10% FBS. Subsequently, samples were filtered through a 100 μm strainer, washed with 0.5% bovine sera-albumin (BSA) in PBS and submitted to Fc receptor blocking with Mouse BD Fc Block (BD Biosciences, Cat# 553141) according to the manufacturer's instructions. The following primary antibodies were added directly to the blocked samples and incubated for 30 min at 4 °C: CD45-PerCP (BD Biosciences, Cat# 557235 1:100), CD11b-FITC (BD Biosciences, Cat# 553310 1:100), F4/80-PE (BD Biosciences, Cat# 563899 1:50), Ly6C-APC (BioLegend, Cat# 128003 1:25) and MHCII-PE-Cy7 (BioLegend, Cat# 116419 1:100). Samples were washed and incubated with 0.25 μg ml⁻¹ 4,6-diamidino-2-phenylindole for 5 min at room temperature. Data were acquired in BD FACSAriaIIu. The gating strategy was as follows: CD45⁺→doublet discrimination (FSC-H, FSC-W)→dead cell exclusion→CD11b⁺ F4/80⁺→morphology (SSC-A, FSC-A)→MHCII^highLy6C⁻ (Supplementary Fig. 8). Data were analysed using FlowJo v10 software.

Monnerat G., Alarcón M.L., Vasconcellos L.R., Hochman-Mendez C., Brasil G., Bassani R.A., Casis O., Malan D., Travassos L.H., Sepúlveda M., Burgos J.I., Vila-Petroff M., Dutra F.F., Bozza M.T., Paiva C.N., Carvalho A.B., Bonomo A., Fleischmann B.K., de Carvalho A.C, & Medei E. (2016). Macrophage-dependent IL-1β production induces cardiac arrhythmias in diabetic mice. Nature Communications, 7, 13344.

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Multiparameter Flow Cytometry Analysis

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The erythrocyte-depleted single cell suspension from BM, inguinal lymph nodes and tumor (as described in Cell Sorting) were incubated with anti-FcγR antibody, followed by incubation with fluorescently labeled antibodies. Ly6G-PE, Ly6C-PerCP/Cy5.5, CD11b-FITC, CD4-PE/Cy7, and MHCII- PE/Cy7 were purchased from Biolegend. Gr-1-PE, Foxp3-FITC, Granzyme B-PE, MHCII-PE, CD11b-PerCP/Cy5.5, LAMP2-eF660, and CD8-AF647 were purchased from eBioscience. PD-1-APC, CCR5-APC and CCR3-PE were purchased from Myltenyi Biotec. Purified anti-CCL5 (Peprotech), secondary APC-conjugated goat anti-rabbit (Columbia Bioscience), iNOS-FITC (BD Bioscience) and CCR2-APC (R&D), CCR1 (R&D) were also acquired from different commercial sources. Data acquisition was performed using FACScan (Becton Dickinson) and analyzed via FlowJo (Tree Star, Inc).

Ban Y., Mai J., Li X., Mitchell-Flack M., Zhang T., Zhang L., Chouchane L., Ferrari M., Shen H, & Ma X. (2017). Targeting autocrine CCL5-CCR5 axis reprograms immunosuppressive myeloid cells and reinvigorates antitumor immunity. Cancer research, 77(11), 2857-2868.

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Multiparameter Flow Cytometry Analysis of Myeloid Cells

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Bone marrow-derived mEOSs were stained with anti-mouse CD45-APC-Cy7 (BioLegend); CD11b-Pe-Cy7 (BD Biosciences, San Jose, CA, USA); CCR3-Pe-Cy7 (BioLegend); CD44-Pe-Cy7 (BioLegend); CD62-L-Pe-Cy7 (BD Biosciences); Siglec-F-APC-Cy7 (BD Biosciences); CD11c-Pe-Cy7 (BioLegend); and MHCII-Pe-Cy7 (BioLegend). Airway mEOSs were stained with CCR3-Pe-Cy7, CD45-APC-Cy7 and NK1.1-biotin (Ablab, Vancouver, Canada); CD90.2-biotin (BioLegend); CD19-biotin (BioLegend); Siglec-F-BV711 (BD Biosciences); CD11c-Pacific Blue (BioLegend); and Ly-6G-PE (BioLegend). Cells were analyzed by using a BD LSRFortessa cytometer (BD Biosciences) and FlowJo software V10 (BD, Franklin Lakes, NJ, USA).

Archambault A.S., Brassard J., Bernatchez É., Martin C., Di Marzo V., Laviolette M., Boulet L.P., Blanchet M.R, & Flamand N. (2022). Human and Mouse Eosinophils Differ in Their Ability to Biosynthesize Eicosanoids, Docosanoids, the Endocannabinoid 2-Arachidonoyl-glycerol and Its Congeners. Cells, 11(1), 141.

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Bone Marrow-Derived Dendritic Cell Isolation and Characterization

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The legs of C57BL/6 male mice (6-8 weeks old) were separated. The bone marrow was flushed out with RPMI-1640 by 1 mL syringe and centrifuged at 1500 rpm for 3 min. Then cells were cultured in RPMI-1640 containing 10% FBS, 1% penicillin-streptomycin and 20 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) (PeproTech, UK) with 5% CO₂ at 37°C. Half of the medium was removed on day 3 and an additional culture medium with GM-CSF (20 ng/mL) was added. BMDCs were harvested on day 6-8 and transferred to a 12-well plate at a density of 2 × 10⁵ cells per well. Cells were treated with SMVs (20 μg), OMVs (2 μg), and HMVs (20 μg) with equivalent protein for 24 h, respectively. PBS-treated cells were used as blank control. Cells were then collected, washed, and blocked with anti-mouse CD16/32 (BioLegend). For specific labeling, cells were stained with anti-mouse antibody against CD11c-FITC (N418, BioLegend), CD11b-Percp/Cy5.5 (M1/70, BioLegend), CD80-PE (16-10A1, eBioscience), CD86-APC (24F, BioLegend), H-2K^b/H-2D^b (MHC-I)-PE/Cy7 (28-8-6, BioLegend) and I-A/I-E (MHC-II) -PE/Cy7 (M5/114.15.2, BioLegend) in 0.5% bovine serum albumin (BSA) dissolved in PBS on ice for 40 min. After washing with PBS for 3 times, cells were analyzed by flow cytometry.

Zhang M., Wang L., Liu J, & Pang Y. (2022). Envelope virus-mimetic nanovaccines by hybridizing bioengineered cell membranes with bacterial vesicles. iScience, 25(6), 104490.

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