Peritoneal exudate cells from three mice were pooled as one sample and used for analysing immune cell profiles by flow cytometry. A total of 15 mice were used for each group (n = 5). Briefly, the peritoneal lavages were centrifuged to collect peritoneal exudate cells. After lysing red blood cells by 1x RBC Lysis Buffer (BioLegend), an equal number of cells from each group were incubated at room temperature for 20 min with Zombie Aqua™ Fixable Viability dye (BioLegend) and blocked on ice for 20 min with Fc Block anti-CD16/CD32 (Thermo Fisher). Then cells were stained with fluorochrome-conjugated monoclonal antibodies (Supplementary Data 1 ) for 1 h. Samples were acquired with the Attune NxT Acoustic Focusing Cytometer using Attune NxT software (Invitrogen), and data were analysed with FlowJo v10.4. For analysis, only singlets (determined by forward scatter height vs. area) and live cells (Zombie Aqua negative) were used. Gating strategy for flow cytometry is shown in Supplementary Fig. 8 .
Fc block anti cd16 cd32
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fc block (anti-CD16/CD32) is a laboratory product designed to block Fc receptors, specifically CD16 and CD32, on cells. This helps reduce non-specific binding of antibodies during immunological experiments. The Fc block is a useful tool for researchers working with flow cytometry, cell-based assays, and other applications where selective Fc receptor blockade is required.
Automatically generated - may contain errors
Lab products found in correlation
Lsr fortessa instrument, by BD (3 mentions)
Dnase 1, by Roche (2 mentions)
Collagenase d, by Roche (2 mentions)
Attune nxt acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Zombie aqua fixable viability dye, by BioLegend (1 mentions)
Attune nxt software, by Thermo Fisher Scientific (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Intracellular fixation permeabilization buffer set, by Thermo Fisher Scientific (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Ifn γ xmg1.2, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm fixation permeabilisation solution kit, by BD (1 mentions)
Il17a 17b7, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Sorp lsr 2, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
11 protocols using fc block anti cd16 cd32
1
Profiling Peritoneal Immune Cells by Flow Cytometry
2
Immune Cell Profiling by Flow Cytometry
3
Flow Cytometry Immunophenotyping Protocol
4
Immunophenotyping of Murine Leukocytes
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Blood was collected from each mouse every week. After plasma isolation and red blood cell lysis, leukocytes were incubated (15 min, 4 °C) with FC Block (anti-CD16/CD32, eBioscience, San Diego, CA, USA) to avoid non-specific binding. After washing, cells were stained for 30 min with extracellular antibodies (anti-CD3, -CD4, -CD8, -CD25, -CD44, -CD62L antibodies, Supplementary Table S2 ). After fixation and permeabilization (FoxP3/Transcription factor staining buffer set, eBioscience), cells were incubated for 30 min with intracellular antibodies to identify regulatory T cells (anti-FoxP3 antibodies, Supplementary Table S2 ). Expression of the various cell markers was determined using a flow cytometer (BD LSRFortessa, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo v10.3 (FlowJo, LLC, Ashland, OR, USA). The gating strategy is described in Supplementary Figure S1 .
5
Intranasal Delivery of DQ-OVA and fmOMV
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DQ™ Ovalbumin (DQ-OVA) (40 μg/head; ThermoFisher Scientific, MA) was delivered intranasally into the lungs in the presence or absence of fmOMV (3 μg/head). After 24 h, mediastinal lymph node (mLN) cells were resuspended in FACS buffer (PBS containing 0.1% bovine serum albumin and 0.01% sodium azide) and incubated with Fc-block (anti-CD16/CD32; eBioscience, CA). After washing, the cells were stained with fluorescence dye-conjugated anti-CD11b, CD11c, Gr-1, CD80, and CD103 antibodies (eBioscience). Samples were acquired on Gallios™ (Beckman Coulter, CA) and data were analyzed using FlowJo software (Tree Star, OH).
6
Flow Cytometric Isolation of Joint Cells
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Harvested joints were digested in collagenase/dispase with DNase and shredded with rat-tooth forceps to isolate cells before staining them for flow cytometry as described (22 (link)). About 5x105 cells were incubated in a 96-well U-bottom plate with Fc block (anti-CD16/CD32; eBioscience, San Diego, CA) then surface stained as indicated with the following antibodies: CD45.2 PE, F4/80 APC, Ly-6G PE-Cy7, and Ly-6C FITC (all from eBioscience). Cells were then washed and fixed in 4% paraformaldehyde for 15 minutes. For each sample 50,000 events were analyzed using a BD LSRFortessa X-20 flow cytometer and data analysis was performed using FlowJo 10.8.1 software.
7
Multiparameter Flow Cytometry for Immune Cell Profiling
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For surface staining, cells were first incubated with Fc block (anti-CD16/CD32, eBioscience) to minimize nonspecific antibody binding, and then stained for 20 min in phosphate-buffered saline / 0.1% bovine serum albumin / 5 mM EDTA buffer with a fixable viability dye and a combination of the following antibodies: CD11b (M1/70), CD11c (N418), MHCII (M5/114.15.2), CD64 (X54-5/7.1) (all from BioLegend, London, UK) and CD45 (30-F11) (from BD Biosciences, Oxford, UK). Following surface staining, cells were fixed with PFA 2%, permeabilized with saponin 0.05% and incubated with an anti-IL-10 APC antibody (clone JES5-16E3, eBioscience) at dilution 1/50 for 20 min. All cells were analyzed on a Fortessa (BD Biosciences) and analysis was performed using FlowJo software (Tree Star, Ashland, OR).
8
Monocyte Identification and Chimerism Analysis
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Erythrocytes were lysed using lysing buffer (BD), and cells were preblocked with anti-CD16/CD32 (Fc Block; eBioscience). Cells were stained on ice for 20 min with combinations of anti-CD45 (APC-Cy7; eBioscience), anti-Ly6C (PerCP-Cy5.5; eBioscience), anti-CD11b (APC; BD), biotinylated anti-CD115 (followed by secondary staining with streptavidin-PE-Cy7; eBioscience), and anti-CD43 (PE; BD) as positive markers and FITC-conjugated lineage markers (CD4, CD19, Ly6G, and Nk1.1; BD) as dump markers for monocytes. For evaluating the reconstitution of chimeric mice, blood cells were stained with anti-CD45.1 (FITC; BD) and anti-CD45.2 (PE-Cy7; BD). Flow cytometry was performed on a FACSCanto II flow cytometer (BD), and FACS data were analyzed with FlowJo Software (Tree Star).
9
Tumor Dissociation and Single-Cell Analysis
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Tumors were cut into small fragments (about 1 mm3) and digested for 1 h 30 min at 37 °C on a rocking plate in HBSS medium containing 5% fetal bovine serum, 1 mg/mL collagenase D (Roche), and 200 U/mL DNase I (Roche). After addition of 5 mM EDTA to block collagenase D activity, the cell suspension was rinsed with PBS and tissue debris eliminated by passing through a 70-μm nylon mesh. Single-cell suspensions were incubated for 20 min at 4 °C with anti-CD16/CD32 Fc Block (eBioscience, San Diego, CA, USA) in PBS containing 1% FCS, 1 mM EDTA, and 0.1% NaN3 (FACS buffer) and stained for 30 min at 4 °C with a mixture of antibodies in FACS buffer. Antibodies recognizing CD45 (47-0451 and 17-0451-83), NK1.1 (12-5941-82), F4/80 (BM8), and CD3 (17-0032-82) were from eBioscience, and CD11b (550993 and 553311), CD11c (550261), Gr1 (552093), and B220 (RA3-6B2) from BD Pharmingen. Tumoral cell lines were also stained with an anti-mouse CCRL2 (clone BZ2E3, BD Pharmingen). Flow cytometry analysis was performed on an LSRFortessa instrument (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using the FlowJo software.
10
Tumor Dissociation and Immune Cell Analysis
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Tumors were cut into small fragments (about 1 mm3) and digested in HBSS medium containing 5% fetal bovine serum, 1 mg/mL collagenase D (Roche), and 200 U/mL DNAse I (Roche) for 1 h 30 at 37°C on a rocking plate. collagenase D activity was blocked by the addition of 5 mM EDTA, and the cell suspension was rinsed with PBS and tissue debris removed by filtering through a 70-μm nylon mesh. Single-cell suspensions were incubated for 30 min at 4°C with anti-CD16/CD32 Fc Block (eBioscience, 14-0161-86) and a mixture of antibodies in FACS buffer (PBS containing 1% FCS, 1 mM EDTA and 0.1% NaN3). Flow cytometry analysis was performed on a LSRFortessa instrument (BD Biosciences) and analyzed using the FlowJo software. The antibodies used were directed to CD45 (47-0451 and 17-0451-83), NK1.1 (12-5941-82), CD3 (17-0032-82), CD4 (48-0041) from eBioscience, and CD8 (551162), CD11b (550993 and 553311), CD11c (550261) and Gr1 (552093) from BD Pharmingen.
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