Cells were washed and blocked with FcR Blocking Reagent (Miltenyi Biotec 130092-575) and stained for cell-surface markers including Live/Dead (eBioscience 650865-14), CD45 (BD Biosciences 563891), CD3 (BD Biosciences 560527), CD4 (BD Biosciences 563331), CD8 (BD Biosciences 563152), NK1.1 (BD Biosciences 562921), F4/80 (BioLegend 123113), CD11b (BioLegend 101222), MHC II (isotype control 400627; BioLegend 107619), CD206 (BioLegend 141708), CD11c (BD Biosciences 564079), Ly6C (BD Biosciences 562728), Ly6G (BD Biosciences 563005), CD80 (BD Biosciences 553769), and CD86 (BD Biosciences 558703). Cells were permeabilized and stained for intracellular IFNγ (isotype control 554686; BD Biosciences 554413). Flow cytometry acquisition was performed on an LSR II cytometer (BD Biosciences), and data were analyzed using FlowJo software version 10.2.
Mhcii
Manufactured by BioLegend
Sourced in United States
MHCII is a laboratory product used in the study of major histocompatibility complex class II proteins. It functions in the presentation of peptide antigens to T helper cells, which is a crucial step in the adaptive immune response.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by BioLegend (38 mentions)
F4 80, by BioLegend (23 mentions)
Cd206, by BioLegend (7 mentions)
Cd103, by BioLegend (7 mentions)
Lsrii flow cytometer, by BD (7 mentions)
Mhc 1, by BioLegend (6 mentions)
Cd62l, by BioLegend (5 mentions)
F4 80, by Thermo Fisher Scientific (5 mentions)
Siglec f, by BD (5 mentions)
Lsrfortessa x 20, by BD (5 mentions)
Facscelesta, by BD (5 mentions)
Fixable viability dye, by Thermo Fisher Scientific (4 mentions)
Fcr blocking antibody, by BioLegend (4 mentions)
F4 80, by BD (4 mentions)
Cd11b, by Thermo Fisher Scientific (4 mentions)
Lsrfortessa, by BD (4 mentions)
Propidium iodide, by Thermo Fisher Scientific (4 mentions)
Lsrii cytometer, by BD (3 mentions)
Granzyme b, by BioLegend (3 mentions)
Facsdiva software, by BD (3 mentions)
89 protocols using mhcii
1
Comprehensive Immune Profiling by Flow Cytometry
2
Immune Cell Activation Analysis
3
OT1 TCR Transgenic Mouse Protocol
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All experiments were performed with 5–10 week old C57BL/6 mice of either sex, purchased from Jackson Laboratories. OT1 are a TCR transgenic mouse specific for the SIINFEKL peptide derived from ovalbumin (amino acids 257–254) in the context of H-2Kb and were bred and housed at the University of Colorado vivarium. All animal protocols were approved by the Institute of Animal Care and Use Committees of the University of Colorado. Antibodies used for depletion, anti-CD8α (clone 53–6.72) and anti-CD8β (clone 53–5.8), were purchased from Bio X Cell and/or made in house. Fluorochrome-conjugated antibodies used for flow cytometry include CD8α, CD8β, B220, CD44, CD62L, MHC-II, CD45.1, CD45.2, CD122, CD127, Eomesodermin and KLRG1 all purchased from Biolegend and Goat anti-Rat IgG from Jackson Immunoresearch Laboratories. Fluorochrome-conjugated antibodies used for microscopy include MOMA-1 (BioRad), IgM (in house), and CD3ε and CD45.1 (Biolegend). SIINFEKL and SIYNFEKL peptide used for in vitro stimulation or in vivo vaccination was synthesized by the University of Colorado Protein Production Shared Resource facility.
4
Immune Cell Phenotyping by Flow Cytometry
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Flow cytometry analyses were performed on a BD Fortessa Analyzer (BD Biosciences) and the data was analyzed with FACSDiva and FlowJo 10 software. Antibodies used were purchased from BD Bioscience [CD11b (clone M1/70), CD11c (clone N418), CD4 (clone RM4-5), CD69 (clone H1.2F3)] or from Biolegend [CD40 (clone 3-/-23, isotype control rat IgG2a), CD80 (clone 16-10A1, isotype control hamster IgG), CD86 (clone GL1, isotype control rat IgG2a), MHCII (clone M5/114.15.2, isotype control rat IgG2b), CD44 (clone IM7)].
5
Immunohistochemistry of Neuroinflammation Markers
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Protein extraction and Western blot analysis were performed as described (Schock et al., 2008 (link)). A custom rabbit antibody against IRF2BP2 was described previously (Teng et al., 2011 (link)). Cryostat sections (20 μm) were subjected to cresyl violet staining and immunofluorescence. Immunofluorescence images were acquired on a Zeiss Z1 fluorescent microscope. Primary antibodies used and their dilutions are: MHCII (Biolegend #107602, anti-rat 500× dilution), CD68 (Santa Cruz, sc70761, anti-mouse, 500×), CD206 (Santa Cruz, sc34577, anti-goat, 500×), Iba1 (WAKO, #019-19741, anti-rat, 500×), NeuN (Millipore MAB377, anti-mouse, 500×), and GFAP (Santa Cruz, sc170, anti-goat, 500×). Cy2-, cy3-, cy5-conjugated secondary antibodies (Jackson Labs) were used at 1000× dilution. For immunofluorescence images, three independent fields at 20× magnification from six sections were imaged and CD206+ and CD68+ cells counted using imageJ.
6
Isolation of Murine Immune Cell Subsets
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Pancreata and spleens were harvested at indicated time points and mechanically disrupted in RPMI. Pancreata were then subjected to enzymatic digestion using RPMI + 10%FBS + 3.6 mg/mL collagenase D (Roche, Basel, Switzerland). Red blood cells from both pancreata and spleens were lysed using red blood cell lysing solution (eBioscience) and the resultant suspension was filtered using a 70-µm cell strainer to remove any resultant debris. Cells were then stained for intracellular and surface antigens as previously described (Surana et al 2014 (link)). The following antibodies were used for surface antigen staining:CD45 (Biolegend; clone 30-F11), CD4 (eBioscience; clone RM4-5), CD8a (Biolegend; clone 53-6.7), CD3 (Biolegend; clone 145-2C11), F4/80 (Biolegend; clone BM8), MHCII (Biolegend; cloneM5/114.15.2), Gr1(Biolegend; clone RB6-8C5), CD11b (Biolegend; clone M1/70), CD11c (Biolegend; clone N418), CD19 (BD Bioscience; clone1D3). In order to isolate CD45+CD3+CD8+ CTLs and CD45+CD3−F4/80+ macrophages, CD45+ leukocytes were first divided into CD3+ or CD3− fractions, and the CD8+CD4− subpopulation among CD45+CD3+ cells and the F4/80+ subpopulation within CD45+CD3− cells were collected using BD FACSAria II (BD Biosciences).
7
Identification and Sorting of M1 and M2 Macrophages
8
Quantification of Lung Immune Cells
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A fixed volume of total lung cells after collagenase digest were stained for major immune cell populations in the presence of Fc block (anti CD16/CD32, eBioscience clone 93) and LIVE/DEAD™ Fixable Yellow Dead Cell Stain at 4°C for 30 mins, using combinations of anti-CD45 (eBioscience clone 30-F11), CD11b (Biolegend clone M1/70), CD11c (Biolegend clone N418), MHCII (Biolegend clone M5/114.15.2), CD24 (BD Biosciences clone M1/69), Siglec-F (BD Biosciences clone E50-2440), Ly6G (Biolegend clone 1A8), CD64 (Biolegend clone X54-5/7.1) (Myeloid cells); anti-CD45, CD11b, CD11c, MHCII, CD64, F4/80 (eBioscience clone BM8), MerTK (eBioscience clone DS5MMER) (macrophage); anti-CD45, EpCAM (eBioscience G8.8), CD31 (eBioscience clone 390) (Epithelial cells). Cells were then washed and resuspended in PBS with 0.2% BSA and a fixed volume run on an Attune flow cytometer to allow quantification of immune cells. EdU was detected using a Click-it EdU imaging kit (Fisher) as per manufacturer's instructions after fixation in 10% formalin (15 mins) and permeabilization in 0.07% saponin, with additional azide from Click Chemistry Tools.
9
Flow Cytometric Immune Profiling
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Murine cells were incubated with a Ghost Dye Red 780 (Tonbo Biosciences) viability marker and anti-mouse CD16/32 (Tonbo Biosciences) in FACS buffer for 30 minutes on ice to block murine Fc receptors. Cells were then stained with the following fluorophore-conjugated anti-mouse monoclonal antibodies: CD45, CD3, CD4, CD8, ICOS [C398.4A], CD11c, CD11b, GR1 [RB6-8C5], F4-80, CD206 [C068C2], B7-1, MHC-I [34-1-2S & 28-8-6], MHC-II [M5/114.15.2], PD-1, Tim3, PD-L1, and B7x [HMH4-5G1] (all from Biolegend); and SPSYVYHQF/H-2Ld Alexa-647 conjugated tetramer [NIH Tetramer Core Facility]. All antibodies were stained for an additional 45 minutes on ice. Human cells were incubated with the Ghost Dye Red 780 viability marker for 30 minutes on ice and immediately stained with the following fluorophore-conjugated anti-human monoclonal antibodies: MHC-I, MHC-II, PD-L1, and B7x [MIH43] (all from Biolegend) on ice for 45 minutes.
10
Isolation and Characterization of Brain Leukocytes
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Leukocytes were isolated from whole brains after digestion in 0.05% collagenase A (Sigma-Aldrich, #C0130) and 10 mg/ml DNase I (Sigma-Aldrich, #D4527), then purified via centrifugation in 37% isotonic Percoll (VWR, # 89428-524) as described [38 (link)]. Isolated leukocytes from brains were stained as described [39 (link)] with fluorescently conjugated antibodies to CD3e (Biolegend, clone 17A2), CD44 (Biolegend, clone IM7), CD19 (Biolegend, clone 6D5), CD107a (Biolegend, clone 1D4B), CD8a (Biolegend, clone 53-6.7), CD4 (Biolegend, clone RM4-5), CD45.2 (Biolegend, clone 104), MHC-II (Biolegend, clone M5/114.15.2), NK1.1 (Biolegend, clone PK136), CD11c (Biolegend, clone N418), F4/80 (Biolegend, clone BM8), CD11b (Biolegend, clone M1/70), Ly6G (Biolegend, clone 1A8), Ly6C (Biolegend, clone HK1.4), CD80 (Biolegend, clone 16-10A1), and Zombie NIR (Biolegend, 423105). Data collection and analysis were performed using an Cytek Northern Lights Cytometer and FlowJo software (Treestar).
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