Anti cd16 32 antibody

Cells were incubated with an Fc receptor blocker, anti-CD16/32 antibody (2 μg ml⁻¹; eBioscience, Cheshire, UK). After washing, cells were incubated with FITC-anti-CD45 (eBioscience), Pacific Blue-anti-MHCII (BioLegend, London, UK), APC-anti-F4/80 (eBioscience), Alex Fluor 700-anti-CD11c (eBioscience), APC-Cy7-anti-11b (BD) and PerCP/Cy5.5-anti-CD103 (BioLegend) antibodies. For assessing intracellular cytokine expression, cells were labelled with FITC-anti-CD3 (BD) and PerCP-anti-CD4 (BD) antibodies, fixed and permeabilised using FOXP3 Fix/Perm buffer set (BioLegend) followed by staining with PE/Cy7-anti-CD25, Alexa Fluor 647-anti-FOXP3, BV-421-anti-IFN-γ, PE-IL-4 and BV-605 anti-IL-17 antibodies (all from BioLegend). Cells were acquired by flow cytometry on the BD LSRII (BD, Oxford, UK) and the data were analysed using FlowJo flow cytometry software (Tree Star, Ashland, OR, USA). The expression of P2X7R in CMT-93 cells was assessed by intracellular staining of P2X7R using FITC-conjugated rat anti-mouse P2X7R antibody (Aviva System Biology, San Diego, CA, USA) and FITC-conjugated rat IgG1 (BD) was used as isotype control.

Cells at (1 × 10⁶/ml) were collected and re-suspended in 300 μl FACS buffer. The cell suspension was then stained for flow cytometry analysis. Fc receptors were blocked using an anti-CD16/32 antibody (2 μg/ml), (eBioscience, Cheshire, UK). The cells were then washed and stained with FITC-anti-CD45 (1 μg/ml), AlexaFluor700-anti CD11c (1 μg/ml), APC-anti-F4/80(1 μl/ml), PE-antiCD86, (2 μg/ml), (all eBioscience), and pacific blue-anti-MHCII (1 μg/ml, BioLegend, London, UK), APC-cy7-anti-CD11b (1 μg/ml, BD, Plymouth, UK), PercP/Cy5.5-anti-CD103 (2 μg/ml) antibodies, and V510 (viability stain, 1 μg/ml).
Single stain controls were prepared using beads or spleen cells, negative controls were prepared using unstained cells. Cells were acquired by flow cytometry on the BD LSRII (BD) and the data were analyzed and visualized using FlowJo™ flow cytometry software (Ashland, OR: Becton, Dickinson and Company; 2019, Berkshire, UK; https://www.flowjo.com/).

Cells derived from in vitro mouse PSC hematopoietic differentiation and the in vivo PB and BM of m-NSG mice were harvested and suspended in PBS with 2% FBS. Before antibody incubation, the cells were blocked with an anti-CD16/32 antibody (eBioscience). The following antibodies were used for analysis: Alexa Fluor 700 (AF700)-conjugated anti-Flk1 (eBioscience), allophycocyanin (APC)-conjugated anti-TIE2 (eBioscience), APC-Cy7-conjugated anti-c-kit (eBioscience), AF700-conjugated anti-Lineage (eBioscience), APC-conjugated anti-Sca-1 (eBioscience), peridinin-chlorophyll protein (PerCP)-eFluor® 710-conjugated anti-CD201 (eBioscience), APC-Cy7-conjugated anti-TER119 (eBioscience), PerCP-Cy5.5-conjugated anti-CD45.1 (eBioscience), and PE-Cy7-conjugated anti-CD45.2 (eBioscience). The following antibodies were used for sorting: APC-Cy7-conjugated anti-c-kit (eBioscience), eFluor450-conjugated anti-Lineage (eBioscience), and phycoerythrin (PE)-conjugated anti-CD201 (eBioscience). Samples were measured by BD Fortessa, and cells were sorted by BD Aria II. The data were analyzed using FlowJo Version 10 software.

The TA muscles of the right and left limbs were collected on days 0, 4, 7, and 15 after injury, combined or not with electroporation with uP-mGM (100 μg/100 μL PBS), as described above. Muscles were incubated with 0.2% collagenase 1A solution (Sigma Aldrich) at 30 °C/45 min. Collagenase activity was blocked by the addition of 5 mL DMEM:FBS (1:1). The samples were filtered in cell strainer 70 and 40 μm.
Isolated cells were washed and resuspended with 0.5% PBS-BSA and preincubated with anti-CD16/32 antibody (eBioscience). Cells were incubated at 4 °C/30 min with the following antibodies: eFluor 450 anti-CD45 (clone 30-F11, eBioscience, catalog number 48-0451-82), FITC anti-F4/80 (clone BM8, eBioscience, catalog number 11-4801-82), APC-eFluor 780 anti-MHCII (clone M5/114.15.2, eBioscience, catalog number 47-5321-82), and PE anti-CD206 (clone C068C2, BioLegend, catalog number 141706). Dead cells were excluded by Fixable Viability Dye eFluor 506 (eBioscience) labeling and fixed in 1% buffered PFA for 15 min. Flow cytometry was performed using FACSCanto II (4.2.2) (BD Bioscience), and data were analyzed using FlowJo software (version 9.2). In this study, CD45+F4/80+MHCII+CD206− macrophages were considered as M1-like [22 (link)–24 (link)] and CD45+F4/80+MHCII−CD206+ as M2-like [23 –25 (link)].

First, BALF cells were washed in PBS containing 0.5% BSA and 0.01% NaN₃ and counted. To avoid nonspecific binding cells were incubated with an unlabeled anti-CD16/32 antibody (AB_467133) before adding a mix containing anti-Gr-1 FITC − (AB_465314), anti-F4/80 PE − (AB_465923), anti-Cd11c APC − (AB_469346), anti-CD3e PE-Cy7 (AB_469572) -labeled antibodies (eBioscience, San Diego, CA). Flow cytometry was performed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo v10 (TreeStar, Ashland, OR) software. Different cell types were identified with the aid of differential marker expression (CD3/B220 + CD11c − Lymphocytes, F4/80 + CD11c + macrophages and Gr-1 + CD3/B220 − CD11c − neutrophils) and via forward and side scatter positioning.