Antibodies to the following were used on a FACS Calibur or LSR II (BD Biosciences, San Jose, CA): fluorophore-labeled CD45, CD11c, Ly6C (Biolegend), CD19, F4/80, CD11b, Ly6G (eBiosciences), MHCII (BD Biosciences). The following were assessed by ELISA: IFNγ (BD Biosciences), IL6, IL12p40, IL13, IL17 (eBiosciences), IL10, TNF (Biolegend), MIP2 (Peprotech).
Mhcii
Manufactured by BD
Sourced in United States, Germany
MHCII is a type of laboratory equipment used to detect and analyze major histocompatibility complex class II molecules, which play a crucial role in the immune system. The core function of MHCII is to facilitate the presentation of antigenic peptides to T helper cells, enabling the immune system to recognize and respond to foreign pathogens.
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Lab products found in correlation
Cd11b, by BD (17 mentions)
F4 80, by BD (10 mentions)
Lsrfortessa, by BD (5 mentions)
Facscanto 2, by BD (5 mentions)
Ifn γ, by BD (4 mentions)
Cd62l, by BD (4 mentions)
Mhc 1, by BD (4 mentions)
Dnase 1, by Merck Group (4 mentions)
Facscalibur, by BD (3 mentions)
Siglec f, by BD (3 mentions)
Cd103, by BD (3 mentions)
Dnase 1, by Roche (3 mentions)
Facscanto 2 flow cytometer, by BD (3 mentions)
Canto 2, by BD (3 mentions)
F4 80, by Thermo Fisher Scientific (2 mentions)
Cd11b, by Thermo Fisher Scientific (2 mentions)
Fc block 2.4g2, by Thermo Fisher Scientific (2 mentions)
Golgiplug, by BD (2 mentions)
Facscanto 2 cytometer, by BD (2 mentions)
Annexin 5, by BD (2 mentions)
53 protocols using mhcii
1
Multiparametric Flow Cytometry Immune Profiling
2
Comprehensive Lung Cell Isolation Protocol
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Lungs were digested in RPMI 1640-based complete medium containing 1 mg/mL of collagenase (Roche) and 30 μg/mL DNase I (Sigma-Aldrich). Samples were passed through an 18-gauge needle, filtered, stained with the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Life Technologies), blocked with anti-CD16/32, and stained with the indicated antibodies. Antibodies were purchased from eBioscience (CD11b, CD11c, F4/80, Ly6C, Ly6G, CD3, CD8, NK1.1, and IFNAR1), R&D systems (CCR2), and BD Biosciences (MHC-II, CD80, CD86, and CD40). To characterize lacZ expression, the FluoReporter LacZ Flow Cytometry Kit was used (Life Technologies). For T cell proliferation, mice were intraperitoneally given 10 mg/mL EdU one day prior to euthanization. Following surface staining, cells were labeled according to the manufacturer’s protocol. Data were acquired on a LSR II (BD Biosciences) and analyzed with FlowJo software (TreeStar).
3
In Vivo MSC Quantification in Mouse Prostate and Tumors
4
Multiparametric Flow Cytometry Analysis
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Cells were suspended in FACS buffer (PBS, 0.1% heat-inactivated fetal calf serum, 0.01% sodium azide), stained for extracellular markers, and fixed in 4% paraformaldehyde. Ab were obtained from eBioscience (San Diego, CA), Biolegend (San Diego, CA), or BD Biosciences (San Jose, CA): TCRβ (Clone H57-597), CD8α (Clone 53-6.7), CD11c (Clone N418), MHC-II (Clone M5/T14.15.2), CD80 (Clone 16-10A1), CD86 (Clone GL1), CD40 (Clone 1C10). OVA-specific dextramer was purchased from Immudex (Copenhagen, Denmark). Aqua LD stain (Life Technologies, Grand Island, NY) was employed for live dead differentiation. Data was captured using FACSCalibur (BD Biosciences), LSR-II (BD Biosciences), or LSR-Fortessa (BD Biosciences) and analyzed using FlowJo (Treestar, Ashland, OR).
5
Flow Cytometric Analysis of BAL Cells
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For flow cytometric analysis, BAL cells were treated with red blood cell lysis buffer (Sigma Aldrich), and cell numbers and viability were assessed via Trypan blue exclusion using a haemocytometer. BAL cells were incubated with Fc block (2.4G2; eBiosciences), followed by staining with fluorochrome‐conjugated monoclonal antibodies to Ly6C, Ly6G, CD11c, and I‐Ab (MHC‐II; BD Biosciences, USA). Neutrophils (Ly6G+), airway macrophages (CD11c+ I‐Ab low), dendritic cells (DC; CD11c+ I‐Ab high), inflammatory macrophages (Ly6G− Ly6C+) were quantified by flow cytometry, as described previously (Tate et al., 2016 ). Live cells (propidium iodide negative) were analysed using a BD FACS Canto II flow cytometer (BD Biosciences) and FlowJo software (RRID:SCR_008520). Total cell counts were calculated from viable cell counts performed via trypan blue exclusion.
6
Analyzing Mouse Immune Cell Phenotypes
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Mouse BMDCs or splenocytes were washed with PBS. For surface marker extracellular staining, the cells were incubated with anti-mouse CD11c (557,400, BD Biosciences), CD80 (560,526, BD Biosciences), CD86 (552,692, BD Biosciences), MHC-II (562,367, BD Biosciences), CD11b (101,211, Biolegend), CD4 (553,046, BD Biosciences), or CD8 antibodies (551,162, BD Biosciences) at 4 °C for 30 min. For intracellular cytokine staining, the splenocytes were stimulated with DnaJ (10 μg/ml) for 8 h in presence of Golgi plug™ (51-2301KZ, BD Bioscience). The splenocytes were then treated for surface markers (CD4 or CD8), fixed/permeabilised with a Cytofix/Cytoperm solution (51-2090KZ, BD Bioscience) and then stained with anti-IFN-γ (557,735, BD Biosciences) and anti-IL-4 (554,435, BD Biosciences) antibodies at 20–25 °C for 30 min. All events were acquired on a FACSverse flow cytometer and analysed using the FlowJoV software (Tree Star).
To determine cytokine (IFN-γ, TNF-α, IL-1β, IL-10, IL-17a, MCP-1, IL-1α, and IL-6) concentrations in the uterine tissue, multi-analyte flow assay kits (740,446, Biolegend, San Diego, CA, USA) were used as indicated by the manufacturer.
To determine cytokine (IFN-γ, TNF-α, IL-1β, IL-10, IL-17a, MCP-1, IL-1α, and IL-6) concentrations in the uterine tissue, multi-analyte flow assay kits (740,446, Biolegend, San Diego, CA, USA) were used as indicated by the manufacturer.
7
Multiparameter Flow Cytometry of Immune Cells
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Spleen and lymph nodes were isolated, and single-cell suspensions were obtained by crushing the tissues through 70 µm filters. The spleen and blood samples were subjected to red blood cell lysis (red blood cell lysis buffer: 8.4 g of NH4Cl and 0.84 g of NaHCO3 per liter of distilled water). Single cell suspensions were stained with fluorescently labeled antibodies (CD45, CD3, CD4, CD8, F4/80, Ly6C, Ly6G, MHCI, MHCII, CD44, CD62L, CD19, CD11C, NK1.1, all from BD Biosciences, San Diego, CA, USA). Nonspecific binding was prevented by the pre-incubation of the cells with a Fc receptor-blocking antibody CD16/32 (Ebioscience, Vienna, Austria). Flow cytometry was performed on a BD Canto II (BD Biosciences), and analysis was performed using the Flowjo software version 10 (Tree star, Ashland, OR, USA).
8
Comprehensive Immune Cell Profiling by Flow Cytometry
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Flow cytometry were carried out using antibodies listed as follows: CD3e [BD Biosciences, 561824 (PE)], CD4 [Thermo Fisher Scientific, 11-0042-82 (FITC)], CD8a [BioLegend, 100706 (FITC)], CD11b [BD Biosciences, 561098 (PE/Cy7)], CD11c [BioLegend, 117353 (BV510)], CD19 [BD Biosciences, 561736 (PE)], CD25 [Thermo Fisher Scientific, 17-0251-82 (APC)], CD45 [BD Biosciences, 561037 (APC-Cy7)], CD49b [BD Biosciences, 561066 (PE)], CD80 [BioLegend, 104714 (APC)], CD274 [BioLegend, 124315 (BV421)], Ly-6G [BD Biosciences, 561104 (PE)], MHC-II (I-A/I-E) [BD Biosciences, 562363 (PerCP-Cy5.5)], and FOXP3 [BioLegend, 126419 (BV421)]. To determine the relative expression of surface markers, cells were stained in FACS buffer for 30 min at 4 °C. For intracellular staining, FOXP3 staining buffer kit (Thermo Fisher Scientific, Waltham, MA) was used following the manufacturer's instructions. Flow cytometry analyses were conducted on a LSR II instrument (BD Biosciences, Mountain View, CA). Results were obtained from the FACSDiva V 7.0 software (BD Biosciences, Mountain View, CA), and all data were analyzed by FlowJo Version 10.0 software. As for FACS, splenic mononuclear cells were isolated, prepared, and stained as described above, followed by sorting of mregDCs as well as non-mregDCs using BD FACSAria flow cytometer (BD Biosciences, Mountain View, CA).
9
Multiparametric Immune Profiling of Tumor Microenvironment
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In independent experiments, which did not include surgery, mice were sacrificed at day 3, 8 or 16 post LVR01-treatment (days 14, 19 or 27 pti) and tumors or TDLN were removed and prepared to obtain single-cell suspensions. Cells were immunostained at 4 °C in the dark for 30 min with antibodies against B220, CD3, CD4, CD8, CD11b, CD11c, CD19, CD27, CD44, CD49b, CD62L, CD69, CD127, CD152 (CTLA-4), CD197 (CCR7), CD279 (PD-1), F4/80, Ly6G, MHCII, NK1.1 and NKG2D (all reagents from BD Pharmingen, San Diego, CA, USA). Absolute cell numbers were obtained using CountBright absolute counting beads (Invitrogen), according to manufacturer's instructions. Flow cytometry data were acquired on a FACS Canto II cytometer and analysed using FACS Diva software (BD Biosciences, Oxford, UK). Flow cytometry gating strategies are illustrated in Supplementary Fig. 1 .
10
Multicolor Flow Cytometry Analysis of iNKT Cells
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