Lungs were perfused with sterile PBS and digested at 37°C for 1 hour with 625 μg/mL collagenase D (Roche 11088875103) and 75 U/mL DNase I (Sigma D4527). Single cell suspensions stained in PBS + 2% FBS + 0.1% sodium azide in the presence of Fc receptor blocking antibody (BD Pharmingen 553541) and stained with the antibodies against the following mouse markers: CD11b_PerCP-Cy5.5 (BD Pharmingen 550993), CD11c_APC-Cy7 (eBioscience 47-0114), Ly6C_PE (BD Pharmingen 560592), Ly6G_PE-Cy7 (BD Pharmingen 560601), and F4/80_APC (Invitrogen MF48005). The FITC channel was used to determine autofluorescence. Cells were stained for 20 minutes at 4C and then fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 20 minutes at room temperature. Flow cytometry was performed on a FACSCanto II (BD Bioscience) and data was analyzed with FlowJo (Tree Star Inc.). Gating strategies are depicted in Extended Data Fig. 6a .
Fc receptor blocking antibody
Manufactured by BD
Sourced in United Kingdom, United States
The Fc receptor blocking antibody is a laboratory reagent designed to block the interaction between the Fc region of antibodies and Fc receptors. This can be used to study antibody-mediated processes in in vitro or ex vivo experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Dnase 1, by Merck Group (2 mentions)
F4 80 apc, by Thermo Fisher Scientific (2 mentions)
Ly6c pe, by BD (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Cd11b percp cy5, by BD (2 mentions)
Ly6g pe cy7, by BD (2 mentions)
Collagenase d, by Roche (2 mentions)
Cd11c apccy7, by Thermo Fisher Scientific (2 mentions)
Horizon brilliant stain buffer, by BD (1 mentions)
Accutase solution, by Merck Group (1 mentions)
Anti cd3, by BD (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Moflo sorter, by Beckman Coulter (1 mentions)
Streptavidin cy3, by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
8 chamber slide, by BD (1 mentions)
Paraformaldehyde (pfa), by ProSciTech (1 mentions)
Tcs sp confocal microscope, by Leica (1 mentions)
Biotinylated donkey anti sheep antibody, by Jackson ImmunoResearch (1 mentions)
6 protocols using fc receptor blocking antibody
1
Lung Tissue Dissociation and Immune Cell Analysis
2
Profiling 3D-UHU CD Surface Markers
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To profile 3D-UHU cluster of differentiation (CD) cell surface markers, terminally differentiated cultures were dissociated using Accutase solution (Merck, UK). Cells were washed with PBS and filtered (100 µm) to prepare single cell suspensions. BD Horizon Fixable Viability Stain 780 solution (1:1000) was added to the cell suspension and incubated at room temperature (RT) for 15 min. Cells were washed and blocked with blocking buffer plus Fc receptor blocking antibody (Clone Fc1.3216, BD Biosciences, UK). Cells were incubated at RT for 10 min then centrifuged (800 g, 3 min). BD Horizon Brilliant Stain Buffer was added to each sample followed by the following antibodies at the optimised volume per test (Supplementary Table 1
3
Bone Marrow and Spleen Cell Phenotyping
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Cells were extracted from bone marrow and spleen. Isolated cells were incubated with Fc receptor–blocking antibody (BD) for 10 min at 4°C to reduce nonspecific staining. Cells were then stained with anti-B220 (PerCP-Cy5.5), anti-CD43 (APC), anti-IgM (FITC), anti-CD25 (APC), anti-IgD (PE), anti-Gr1 (PE), anti-CD11b (APC), anti-CD21 (FITC), anti-CD23 (PE), anti-CD93 (APC), and anti-CD3 (FITC; BD) for 30 min at 4°C in the dark. For intracellular IgHµ staining, cells were first stained with surface markers, permeabilized and fixed with buffer Perm/Wash (BD), and stained with anti-IgM mu-biotin antibody (Jackson ImmunoResearch Laboratories, Inc.) followed by incubation with streptavidin-PE. Cells were processed in a Gallios flow cytometer (Beckman Coulter), and the data were analyzed using FlowJo software (Tree Star). For cell-sorting experiments, bone marrow cells were incubated with Fc receptor–binding antibody and then stained with anti-B220 (PerCP-Cy5.5), anti-CD43 (APC), and anti-IgM (FITC; BD), under the same conditions. B220+CD43+IgM− cells were isolated on a MoFlo sorter (Beckman Coulter).
4
Lung Tissue Dissociation and Immune Cell Analysis
5
Visualizing CLIC1 and RhoA in BMDCs
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Resting BMDCs or BMDCs 5 min after initiation of synchronised phagocytosis were fixed with 4% paraformaldehyde (Cat #C004, ProSciTech) on 8-chamber slides (Cat #354108, BD Biosciences), then permeabilised with 0.05% saponin (Cat #S4521, Sigma-Aldrich) (Jiang et al., 2012 (link)). After blocking with 2% IgG free BSA (Cat #001-000-161, Jackson ImmunoResearch Labs) and 1 µg/ml Fc receptor blocking antibody (Cat #553142, BD Biosciences), the cells were stained for; CLIC1 and RhoA. Briefly, BMDCs were firstly stained with 1:100 rabbit anti-mouse RhoA antibody then 1:100 donkey anti-rabbit Cy2 antibody (Cat#ab6940, Abcam) followed by an in-house-derived 1:1000 sheep anti-mouse CLIC1 (Jiang et al., 2012 (link)), then 1:100 biotinylated donkey anti-sheep antibody (Cat #713-065-003, Jackson ImmunoResearch Labs) and finally streptavidin Cy3 (Cat#S6402, Sigma-Aldrich) (Jiang et al., 2012 (link)). Confocal images were obtained on a Leica TCS SP confocal microscope (Leica Microsystems, Germany) and processed using ImageJ64 (NIH, imagej.nih.gov/ij/download/).
6
Lung Tissue Dissociation and Flow Cytometry
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Chopped lung tissues were incubated in RPMI-1640 complete medium containing 10% type IV collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA) at 37°C for 90 min and filtered with a 0.45 μm sterile cell strainer. The cells in each tube (1×106 cell density) were blocked with Fc receptor-blocking antibody (BD Biosciences) and then incubated with fluorochrome-labeled surface or cytokine antibodies (BioLegend, San Diego, CA, USA) at 4°C for 30 min. The antibodies used are listed in Supplementary Table 2 . Flow cytometry was performed using a BD LSRFortessa™ X-20 (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo (version 10.8) software (TreeStar, Woodburn, OR, USA).
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