A mouse LP dissociation kit (Miltenyi Biotec) was used to isolate cells from the small intestine according to the manufacturer's protocol, with a modification at the final dissociation step where cells were gently crushed through a 70 μm cell strainer immediately following enzymatic digestion to generate a single cell suspension. Cells were centrifuged for 10 min at 1,500 rpm and the cell pellet was resuspended in PBS containing 0.5% BSA. Cells were counted using a hemocytometer prior to staining. Cells were stained with fixable viability dye eFluor506 (ThermoFisher) and Fc receptors were blocked with anti-mouse CD16/32 (2.4G2; BD Biosciences). Surface staining included rat anti-mouse CD45 (30-F11; BD Biosciences), rat anti-mouse B220 (RA3-6B2; BD Biosciences) and rat anti-mouse Siglec F (E50-2440; BD Biosciences). Cells were fixed and permeabilized using BD CytoFix/CytoPerm (BD Biosciences) and incubated with rat anti-mouse IgA (C10-1; BD Biosciences). Liquid counting beads (BD Biosciences) were added to samples prior to acquiring data on a CytoFLEX flow cytometer (Beckman Coulter) to enable total cell counts of cells falling within IgA+B220−CD45+ live gates according to manufacturer's protocol. Data were analyzed using CytExpert software (Beckman Coulter).
Anti mouse cd16 32 2.4g2
Manufactured by BD
Sourced in United States
Anti-mouse CD16/32 (2.4G2) is a monoclonal antibody that binds to the mouse CD16 and CD32 receptors. It can be used to block Fc receptor-mediated interactions in cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Phalloidin ifluor 405, by Abcam (2 mentions)
P47phox a 7, by Santa Cruz Biotechnology (2 mentions)
Clone x54 5 7, by BioLegend (2 mentions)
Fab639p, by R&D Systems (2 mentions)
Clone rh1, by BioLegend (2 mentions)
Clone m18 2, by BioLegend (2 mentions)
Clone m1 70, by BioLegend (2 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Liquid counting beads, by BD (1 mentions)
Cytexpert software, by Beckman Coulter (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Rat anti mouse cd45 30 f11, by BD (1 mentions)
Rat anti mouse b220 ra3 6b2, by BD (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Tnf α, by BioLegend (1 mentions)
Il 1β, by BioLegend (1 mentions)
Tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Zombie yellow fixable viability kit, by BioLegend (1 mentions)
Flow count beads, by Beckman Coulter (1 mentions)
9 protocols using anti mouse cd16 32 2.4g2
1
Isolation of Intestinal IgA+ B Cells
2
Isolation and Characterization of Peritoneal Cavity Cells
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Collection of PCCs was done as described above. As previously reported (50 ), 5 million to 10 million PCCs per mouse can be obtained from the peritoneal cavity. Among all live cells, 50 to 60% are B cells, 30% are macrophages, and 5 to 10% are T cells. The B cells consist of 65 to 70% B1 cells and 25 to 30% B2 cells. PCCs were resuspended in RPMI 1640 containing 10% FBS supplemented with penicillin and streptomycin (Gibco) and cultured on a 24-well plate with 2 mM d -alanine, TNF-α (10 ng/ml) (BioLegend), IL-1β (10 ng/ml) (BioLegend) for 5 days. These cells were harvested in PBS containing 1% BSA, blocked with FcγR-blocking mAb [anti-mouse CD16/32 (2.4G2), BD Biosciences, 553142] for 10 min, and stained with FITC anti-CD45 (30-F11), APC anti-B220 (RA3-6B2), and PE-IgM (RMM-1) at 4°C for 30 min. After being washed in 1% BSA-PBS, cells were stained with 7-AAD for 5 min before flow cytometry analysis.
3
Neutrophil Localization of EphA2 and p47phox
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To determine protein localization within the neutrophils, the cells were incubated in RPMI 1640 medium containing 2% heat-inactivated mouse serum for 30 min on fibronectin coated coverslips. The cells were then incubated with opsonized C. albicans CAI4-GFP for 30 min. Next, they were fixed with 3% paraformaldehyde, blocked with 10% BSA, followed by anti-mouse CD16/32 (2.4G2; BD Biosciences), and incubated with antibodies against EphA2 (D4A2, #6997; Cell Signaling), and p47phox (A-7; Santa Cruz), followed by an Alexa 488-conjugated mouse anti-rabbit antibody. To visualize F-actin, the cells were also stained with Phalloidin-IFluor 405 (Abcam). The cells were then imaged by confocal microscopy.
4
Tumor-Infiltrating Cell Characterization
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Tumor-infiltrating cells were prepared with a tumor dissociation kit (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. The total number of live cells analyzed was matched between the groups with Flow Count beads (Beckman Coulter, Fullerton, CA, USA). A Zombie Yellow fixable viability kit (BioLegend, San Diego, CA, USA) was used to stain dead cells. The cells were then pretreated with purified anti-mouse CD16/32 (2.4G2) (BD Biosciences, NJ, USA), stained with antibodies, and analyzed with a CytoFLEX (V5-B5-R3 configuration, Beckman Coulter, Fullerton, CA, USA). The following fluorescent-labeled antibodies were purchased from BioLegend (San Diego, CA, USA) and used for analysis: Brilliant Violet 785-conjugated anti-CD45 (30-F11), Brilliant Violet 605-conjugated anti CD11b (M1/70), allophycocyanin (APC)-conjugated anti-F4/80 (BM8), phycoerythrin (PE)-Cyanine7-conjugated anti-CD86 (GL-1), PE-conjugated anti-CD206 (MMR), and fluorescein isothiocyanate (FITC)-conjugated anti-CD49b (HMα2). For all channels, positive and negative cells were gated on the basis of fluorescence minus one controls, and CD86 and CD206 were gated with appropriate isotype controls (Figure S1 ). The data were analyzed with FlowJo software v10.4.
5
Multiparameter Flow Cytometry Profiling
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Single-cell suspensions from in vitro cultures or harvested LNs were analysed by flow cytometry. Cells were washed in flow cytometry staining buffer (PBS with 2% FCS and 0.02% sodium azide) followed by blocking with anti-mouse CD16/32 (2.4G2; BD Bioscience). The following mAbs from BD Biosciences, CD4-PE/V450 (RM4-5), CD80-PE-CF594 (16-10A1), CD40-PE (3/23), eBioscience; CD3-PE-eFluor-610 (145-2C11), CD11b-PerCP-Cy5.5/PE-Cy7 (M1/70), CD11c-PE/PE-Cy7 (N418), MHC-II-FITC/eFluor-450 (M5/114.15.2), Biolegend; PD-L1-PE/APC (10F.9G2), DEC-205-APC (NLDC-145), and Miltenyi Biotec; CD86-FITC/PE (PO3.3) were used at optimally titrated concentrations. For transcription factor staining, cells were fixed and permeabilized with a commercial transcription factor staining kit (eBioscience) according to the manufacturer's instructions, and the cells were stained with the following transcription factors from BD Bioscience—FoxP3-PE/ PE-CF594 (MF23), T-bet-FITC/APC (O4-46), RORγt-BV421 (Q31-378) and eBioscience GATA3-PE (TWAJ). Viable cells were distinguished using LIVE DEAD Aqua (Life Technologies). Populations of interest were gated according to appropriate “fluorescence minus one” controls. Samples were acquired on a CyAn ADP flow cytometer (Beckman Coulter) and were analyzed with FlowJo software (Tree Star).
6
Neutrophil Localization of EphA2 and p47phox
7
Neutrophil Surface Marker Profiling
8
Isolation and Analysis of Liver Leukocytes
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To obtain single cell suspensions, liver tissues were mechanically disrupted, cells pelleted, and resuspended in 7.5 mL PBS and 4.5 mL isotonic Percoll (Sigma-Aldrich), and centrifuged at room temperature for 15 min at 760 × g. Leukocytes and red blood cells (RBCs) were harvested, and RBCs lysed in lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA). Intracellular cytokine staining was performed on leukocytes using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Fc receptor binding was blocked with anti-mouse CD16/32 (2.4G2; BD Pharmingen) before addition of antibodies against mouse CD3 (145-2C11 or 17A2; PE, PerCP or eFluor 450; eBioscience), NK1.1 (PK136; APC; eBioscience and BD Pharmingen), Fzd1 (162531; biotin; R&D Systems), Fzd7 (151143; biotin; R&D Systems), TCRβ (H57-597; BV421; BioLegend), CD11b (M1/70; PE; BD Pharmingen), Ly-6G (1A8; Alexa Fluor 700; BioLegend), Ly-6C (AL-21; PE-Cy7; BD Biosciences), IFN-γ (XMG1.2; FITC; BD Pharmingen), and IL-4 (BVD4-1D11; PE; BD Pharmingen). Streptavidin-PE-Cy7 (BD Pharmingen) was used for detection of biotinylated antibodies. α-GalCer-loaded CD1d tetramer (PE) was generated in-house using a baculovirus expression system, similar to that previously described (26 (link)). Samples were acquired on a BD FACSCanto or Beckman Coulter Gallios and analyzed using FlowJo software (TreeStar, Inc.).
9
Neutrophil Surface Marker Profiling
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Mouse neutrophils were isolated as described above. Purified neutrophils were incubated with anti-mouse CD16/32 (2.4G2; BD Biosciences) blocking antibody, followed by anti-mEphA2 (PE conjugated; FAB639P; R&D Systems), anti-Dectin-1 (PE conjugated; Clone RH1; BioLegend), anti-CD11b (PE conjugated; Clone M1/70; BioLegend), anti-CD18 (PE conjugated; Clone M18/2; BioLegend), anti-CD64 (PE conjugated; Clone X54–5/7.1; BioLegend), and surface expression was measured using flow cytometry. For CD16/32 staining neutrophils were stained with anti-CD16/32 (PE conjugated; Clone 93; BioLegend) without Fc-block.
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