2×105 to 1×106 cells were incubated with near IR LIVE/DEAD fixable cell stain kit according to the manufacturer’s instruction (Life Technologies) followed by anti-mouse CD16/32 Fc block (eBioscience) for 20 min at 4°C. After washing with PBA (PBS containing 1% BSA, 0.1% sodium azide), cells were stained with a customised extracellular antibody panel for 30 min at 4°C in PBA and then fixed for 20 min with 2% paraformaldehyde or kept on ice with 5 μM EDTA prior to sorting. Cells were run on a BD FACS Canto II collecting at least 10,000 events of the target population and analysed using FlowJo (Tree Star, Ashland, OR, US). A BD Influx was used for sorting pure airway and peritoneal macrophages for qPCR analysis. Alveolar macrophages were identified as CD11bloCD11chiF4/80hi and highly auto-fluorescent. Peritoneal macrophages were identified as CD11bhighCD11cloF4/80hi. All lineage markers were purchased from eBioscience (Hatfield, UK) or Biolegend (San Diego, CA, USA). Axl (clone 175128), MerTK (polyclonal goat IgG, biotinylated), Tyro3 (clone 109646) and Gas6 (polyclonal goat IgG, biotinylated) antibodies were from R&D Systems (Abingdon, UK). Axl and Tyro3 antibodies were conjugated to Alexa Fluor-647 dye using a labelling kit (Life Technologies) according to the manufacturer’s instruction.
Fc block anti mouse cd16 32
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fc block anti-mouse CD16/32 is a laboratory equipment product designed to block Fc receptors on mouse cells. It is used to prevent non-specific binding of antibodies during immunological assays or experiments involving mouse cells.
Automatically generated - may contain errors
Lab products found in correlation
Percp cy5.5 labelled anti cd11c n418, by BioLegend (2 mentions)
Labeled anti nk 1.1 pk136, by Thermo Fisher Scientific (2 mentions)
Fitc labeled anti ly6g rb6 8c5, by Thermo Fisher Scientific (2 mentions)
Uniform dyed microspheres, by Bangs Laboratories (2 mentions)
Rm4 5, by BioLegend (2 mentions)
Facscanto 2, by BD (1 mentions)
Alexa fluor 647 dye, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Collagenase a, by Merck Group (1 mentions)
Live dead bv510, by Thermo Fisher Scientific (1 mentions)
Anti f4 80 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd45 apc cyanine7, by BioLegend (1 mentions)
Anti cd11b fitc, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Guava easycyte, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Stabilizing fixative, by BD (1 mentions)
Collagenase d, by Roche (1 mentions)
7 protocols using fc block anti mouse cd16 32
1
Multiparametric Flow Cytometry of Macrophages
2
Lung Immune Cell Profiling Protocol
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The lungs were collected on day 14 after SAL or BLM installation. Briefly, the lungs obtained from each mouse were digested by collagenase A (1 mg/ml; Sigma-Aldrich) for at least 40 min, with shaking at 37°C. Phosphate-buffered saline containing 10% fetal bovine serum was added to inactivate the enzyme, and the solution was passed through a 40-µm cell strainer. Cells (106 cells/ml) obtained from the lungs or BAL were incubated with LIVE/DEAD BV510 according to the manufacturer’s instruction (Life Technologies, Carlsbad, CA, USA) followed by anti-mouse CD16/32 Fc block (eBioscience, San Diego, CA) for 15 min at 4°C. Different cell populations were identified using the following corresponding antibodies: a) Ly6C-FITC, Ly6G-Alexa647, CD11b-Pecy7, CD11c-PECy5, and SinglecF-PE (monocytes, neutrophils, eosinophils, and alveolar macrophages) or b) CD11b-APC, CD11c-PECy5, SinglecF-FITC, CD86 PECy7, and CD206-PE (alveolar macrophage activation). Samples were acquired by BD LSRFortessa X-20 Cell Analyzer (BD Biosciences, San Rose, CA) and then analyzed by FlowJo software (Ashland, OR, USA).
3
Kidney Immune Cell Profiling
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Kidney single-cell suspensions were prepared via mechanical and enzymatic digestion as previously described (23 (link)). Suspensions were incubated with Fc Block anti-mouse CD16/32 (catalog no. 14-0161-85, eBioscience) for 15 min and then treated with anti-CD45-APC/Cyanine7 (catalog no. 103115, BioLegend), anti-CD11b-FITC (553310, BD), anti-F4/80-PE (12-4801-80, eBioscience), anti-CD206-Alexa Fluor 647 (565250, BD), and anti-CD86-eFluor 450 (48-0862-82; eBioscience) for 30 min at 4°C. Cells were washed with PBS, resuspended with 200 μl PBS, and detected using a Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). Data were analyzed using FlowJo software version 10.
4
Lymph Node Single-Cell Analysis Protocol
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Lymph nodes of all mice in each of the 3 groups (vehicle, HT, and OPT3-treated) were removed from all animals on the last day and mashed the same day through a cell strainer, as previously described [55 (link)]. Briefly, single-cell suspensions were transferred to FACS buffer (0.5% N,O-bis (trimethylsilyl)acetamide—BSA, 1% FBS, and Ethylenediaminetetraacetic acid—EDTA 1:50 in DPBS—Thermo Fisher Scientific, Rockford, IL, USA). After blocking with 0.5 mg/mL Fc block anti-mouse CD16/32 (eBioscience, San Diego, CA, USA) for 15 min, the samples were stained with fluorescent-labelled anti-mouse CD3, CD4, and CD8a antibodies (Table 1 —also including their concentrations), for 30 min at 4 °C and protected from light, following the manufacturer’s instructions. Subsequently, cells were washed and resuspended in DPBS, and eFluor450fixable viability dye staining (eBioscience, San Diego, CA, USA) was performed. After washing the cells again, a stabilizing fixative (eBioscience, San Diego, CA, USA) was added. Stained samples were analyzed on a FACS Canto (BD, Germany) and results were extracted using FlowJo Software (FlowJo LLC, Tree Star Inbc., Ashland, OR, USA).
5
Analysis of Lung Immune Cells by Flow Cytometry
6
Naïve, OVA, and Allergic Mouse Nictitating Membrane Analysis
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Nictitating membranes of naïve, OVA stimulated, and allergic mice were dissected. Because of the small amount of tissue, the eyes of five mice each were pooled into one sample for analysis. Tissues were digested with 2 mg/mL collagenase D and 0.1 mg/mL DNAse I (Roche, Germany) in HBSS. Cells were transferred to FACS buffer (0.5% BSA, 1% FBS, EDTA 1:50 in PBS), and blocked with Fc block anti-mouse CD16/32 (eBioscience, Germany). Cells suspensions were incubated with CD11b, CD11c, Ly6G, Ly6C, CD45, CD64 (BioLegend, Germany), MHCII, SiglecF (BD Bioscience, Germany), and F4/80 (eBioscience, Germany) antibodies according to the manufacturer’s instructions for 30 min on ice and protected from light. After washing the cells, the pellet was resuspended in PBS, and eFluor450 fixable viability dye staining was performed (BD Bioscience, Germany). Cells were then washed and fixed in stabilizing fixative (BD Bioscience, Germany). FACS analyses were performed using either an LSR Fortessa (BD Bioscience) or Guava easyCyte (Merck Millipore) cytometer. Raw data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA). Three independent experiments were conducted.
7
Analysis of Lung Immune Cells by Flow Cytometry
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Analysis of cell populations in BALF or single cell suspension of lung homogenate was conducted as before46 (link). Cells were labeled with a combination of phycoerythrin (PE)-labeled anti-NK 1.1 (PK136; eBioscience), fluorescein (FITC)-labeled anti-Ly6G (RB6-8C5; eBioscience), allophycocyanin (APC)-labeled anti-MHCII (M5/114.15.2; eBioscience), PerCP-Cy5.5-labelled anti-CD11c (N418; Biolegend), or phycoerythrin (PE)-labeled CD4 (RM4-5; Biolegend) alone. Neutrophils (PMNs) were defined as Ly6G+/MHCII− , Macrophages as CD11c+/MHCIIlow-mid and Dendritic cells (DCs) as CD11c+/MHCIIhigh. Fc block (anti-mouse CD16/32) was also added to each sample (93; eBioscience). Uniform dyed microspheres (Bangs Laboratories, Inc) were added to calculate the concentration of cellular components. Samples were run on FACS Calibur and analyzed on FlowJo (ver 10.0.6).
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