Six well plates were coated overnight at 4°C with antibody, anti-NK1.1 (PK136), anti-NKp46 (29A1.4) or anti-NKG2D (A10) (eBioscience, San Diego, CA). After sacrifice splenocytes were harvested and red blood cells lysed using ACK lysis buffer (eBioscience, San Diego, CA). 6 × 106 cells were incubated either in an antibody coated, unstimulated or PMA and ionomycin treated well for five hours in the presence of GolgiPlug (BD Biosciences, San Diego, CA). Following incubation Fc receptors were blocked with anti-CD16/CD32 (2.4G2) antibody (BD Biosciences, San Diego, CA). Invitrogen fixable aqua live/dead stain was used according to manufacturer’s instructions for dead cell exclusion. NK1.1 (PK136) (for all stimulation conditions other than samples stimulated with anti-NK1.1 antibody) or NKp46 (for samples stimulated with anti-NK1.1 antibody) and CD3ε (145-2C11) surface markers were stained (eBioscience, San Diego, CA). Cells were fixed and permeabilized using BD Cytofix/Cytoperm™ kit (BD Biosciences, San Diego, CA) according to manufactures instructions. Fc receptors were blocked with anti-CD16/CD32 (2.4G2) antibody. Intracellular staining was performed using an anti-IFNγ (XMG1.2) antibody (eBioscience, San Diego, CA). Cells were analyzed using a BD LSRFortessa cell analyzer (BD Biosciences, San Diego, CA)
Anti cd16 cd32 antibody 2.4g2
Manufactured by BD
Sourced in United States
The Anti-CD16/CD32 antibody (2.4G2) is a laboratory reagent that binds to the Fc gamma receptors CD16 and CD32. It is commonly used to block non-specific binding of antibodies in immunological assays.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (2 mentions)
Lsrfortessa cell analyzer, by BD (2 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pacific blue anti cd45 30 f11, by BioLegend (2 mentions)
Nk1.1 pk136, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Cd3ε 145 2c11, by Thermo Fisher Scientific (1 mentions)
Anti nkg2d a10, by Thermo Fisher Scientific (1 mentions)
Rs2000, by Rad Source (1 mentions)
Facsaria, by BD (1 mentions)
F ova, by Thermo Fisher Scientific (1 mentions)
Cd11c microbeads ultrapure, by Miltenyi Biotec (1 mentions)
Dnase 1, by Roche (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
Collagenase d, by Roche (1 mentions)
Anti allophycocyanin magnetic microbeads, by Miltenyi Biotec (1 mentions)
Facsaria 2, by BD (1 mentions)
Ghost dye violet 510, by Cytek Biosciences (1 mentions)
Anti cd45 30 f11, by BioLegend (1 mentions)
Anti cd11b m1 70, by BioLegend (1 mentions)
11 protocols using anti cd16 cd32 antibody 2.4g2
1
Characterizing Murine NK Cell Activation
2
Bone Marrow Transplantation in Mice
3
In Vivo Cellular Uptake of Fluorescein-Labeled Ovalbumin
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Fluorescein conjugates of ovalbumin (F-OVA, Life Technologies, Carlsbad, CA, USA) were used as the cancer antigen to test its in vivo cellular uptake. First, F-OVA (100 μg) was simply mixed with HMS nanoparticles or HPS microparticles (0.9 mg/100 μL) in saline and subcutaneously injected into the flank of a mouse (C57BL/6J, female, 6 weeks old, CLEA Inc., Tokyo, Japan). The mice were divided into four groups: (1) F-OVA, (2) HMS-F-OVA, (3) HPS-F-OVA, and (4) HMS-HPS-F-OVA. Cells around the injection site were collected on d3 to prepare a single-cell suspension. Non-specific staining was inhibited by anti-CD16/CD32 antibody (2.4G2, BD Pharmingen, San Jose, CA, USA). Then, the cells were stained using anti-mouse CD11c and anti-mouse CD86 antibodies (Biolegend, San Diego, CA, USA) for 30 min. Flow cytometry was performed using FACSAria (BD Bioscience, Franklin Lakes, NJ, USA).
4
Multicolor Flow Cytometry Immune Phenotyping
5
Isolation of Tissue Macrophages and Dendritic Cells
6
Isolating Microglia from 5XFAD Mice
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Ghost dye Violet 510 (1:1,000, Tonbo Biosciences) and anti-CD16/CD32 antibody (2.4G2, 1:100, BD Biosciences) were used to exclude dead cells and to block Fc receptors, respectively. Single-cell suspensions were then stained with anti-CD11b (M1/70, 1:100, Biolegend), anti-CD45 (30-F11, 1:100, Biolegend), and anti-CD11c (N418, 1:50, Biolegend) antibodies for 20 min on ice. Samples were washed with ice-cold FACS buffer, spun down for 5 min at 500 g, and cell pellets were resuspended in 5 mL FACS buffer before sorting on a BD FACS Aria II using the 70-μm nozzle with purity mode and a sorting speed of ~10,000 events per second. After sorting, each sample was spun down, and cell pellets were immediately stored at −80° until further processing.
Due to the small population size of CD11c+ microglia and low yield of microglia by cold isolation in general, two biological samples were prepared for each group. For 9-mo-old 5XFAD mice, each CD11c+ microglial sample was pooled from 22 mice (11 male + 11 female) for a total of 44 mice (22 male + 22 female). For 9-mo-old OPN-KO.5XFAD mice, one CD11c+ microglia sample was pooled from 13 mice (6 male + 7 female) and the other sample was pooled from 14 mice (7 male + 7 female) for a total of 27 mice (13 male +14 female).
Due to the small population size of CD11c+ microglia and low yield of microglia by cold isolation in general, two biological samples were prepared for each group. For 9-mo-old 5XFAD mice, each CD11c+ microglial sample was pooled from 22 mice (11 male + 11 female) for a total of 44 mice (22 male + 22 female). For 9-mo-old OPN-KO.5XFAD mice, one CD11c+ microglia sample was pooled from 13 mice (6 male + 7 female) and the other sample was pooled from 14 mice (7 male + 7 female) for a total of 27 mice (13 male +14 female).
7
Flow Cytometry Analysis of Immune Cells
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The subsets of immune cells in the bone marrow, spleen, and lymph nodes were analyzed with a flow cytometer (FACSCanto II; BD Biosciences, Franklin, NJ, USA). To block FcγR, single-cell suspensions were incubated with the anti-CD16/CD32 antibody (2.4G2; BD Biosciences) on ice for 10 min before staining with the indicated monoclonal antibodies. The following monoclonal antibodies were used in this study: anti-IgM (RMM-1), anti-IgD (11-26c.2a), anti-CD19 (6D5), anti-mouse CD45R/B220 (RA3-6B2), anti-CD43 (S11), anti-CD21/CD35 (7E9), anti-CD23 (B3B4), anti-CD4 (RM4-4), anti-CD8a (53-6.7), and anti-CD25 (PC61, all from BioLegend, San Diego, CA, USA), and anti-CD3ε (eBioscience, San Diego, CA, USA); all antibodies were conjugated with fluorochrome. Dead cells were excluded by 7-aminoactinomycin D (7-AAD; BioLegend) staining. In most samples, a minimum of 3 × 104 events was evaluated, with all data analyzed using FlowJo software (version 9.9.5; BD Biosciences).
8
Multicolor Flow Cytometry Immunophenotyping
9
Isolation of Cardiac Immune Cells
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Single-cell suspensions were prepared as described previously (12 (link), 28 (link)). Briefly, mice were deeply anesthetized and intracardially perfused with 20 ml of ice-cold PBS to eliminate blood cells. The hearts were minced with fine scissors and placed into a cocktail of 1 mg/ml collagenase II (Worthington, Lakewood, NJ, USA), 100 U/ml elastase (Worthington), and 100 U/ml DNase I (Sigma-Aldrich) and shaken at 37°C for 1 h. Tissue samples were then triturated through a 70 μm cell strainer and centrifuged (5 min, 400 g, 4°C). The obtained cells were counted after erythrocyte lysis and washed using PBS for further analysis. For staining, 5 × 106 cells were pre-incubated for 5 min on ice with anti-CD16/CD32 antibody (2.4G2, BD Bioscience, San Jose, CA, USA) to block the non-specific antibody and then stained with directly conjugated antibodies for 30 min at 4°C in the dark in PBS. For intracellular cytokine staining, single-cell suspensions were stimulated with 50 ng/ml (PMA, Sigma-Aldrich), 1 μg/ml ionomycin (Sigma-Aldrich), and Golgi-PlugTM (BD Biosciences) for 4 h. Surface staining was performed first. After fixation and permeabilization using the Cytofix/Cytoperm Soln kit (BD Biosciences), intercellular proteins were stained. All experiments were performed on an Attune NxT flow cytometer (Invitrogen) and analyzed using FlowJo version 10 software.
10
Liver Immune Cell Profiling by Flow Cytometry
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