Whole lungs were removed and chopped with razor blades, incubated with type IV collagenase (Worthington) at 37 °C for 40 min, then homogenized through a 70-µm cell strainer (Falcon). Remaining red blood cells were lysed using 1× red blood cell lysis buffer (BD Biosciences). Cells were stained with Fixable Viability Dye eFluor® 455 (eBioscience). Anti-mouse immunophenotyping antibodies were diluted in FACS buffer (3% FBS, 2 mM EDTA, 1× PBS) to 5 µg per mL along with Fc block (anti-mouse CD16/CD32; 5 µg per mL, BD), and cells were stained for 30 min on ice in three groups (AMφ: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), and Siglec-F (E50-2440; BD); monocyte and neutrophil: Ly-6C (AL-21; BD), Ly-6G (1A8; BD), and CD11b (M1/70; BD); dendritic cell: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), MHC II (M5/114.15.2; BD), and CD103 (M290; BD); NK cells: CD3 (17A2; BD), NK-1.1 (PK136; BD), and CD49b (DX5; BD)). After the staining, cells were washed twice with FACS buffer and then fixed in 2% para-formaldehyde in FACS buffer for 15 min. Cell numbers were counted using AccuCount Fluorescent Particles (Spherotech). All data were collected on an LSR II flow cytometer (BD) and analyzed using FlowJo software.
Anti mouse cd16 cd32 fc block
Manufactured by BD
Sourced in United States
Anti-mouse CD16/CD32 Fc block is a laboratory reagent used to block non-specific Fc receptor binding in immunoassays involving mouse cells or tissues. It helps to minimize the risk of false-positive results by preventing unwanted interactions between Fc receptors and antibodies used in the assay.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (3 mentions)
Cd69 clone h1.2f3, by BD (3 mentions)
Lsrfortessa, by BD (3 mentions)
Efluor 780, by Thermo Fisher Scientific (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Kaluza flow analysis software, by Beckman Coulter (2 mentions)
Cyan adp, by Beckman Coulter (2 mentions)
Collagenase 1, by Merck Group (2 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions)
Collagenase 4, by Worthington (2 mentions)
Collagenase type xi, by Merck Group (2 mentions)
Perm wash, by BD (2 mentions)
Fix perm solution, by BD (2 mentions)
Type 4 bovine pancreatic dnase, by Merck Group (2 mentions)
Granzyme b clone gb12, by Thermo Fisher Scientific (2 mentions)
Lsr 2 digital flow cytometer, by BD (2 mentions)
Ifnγ clone xmg1, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Facsdiva software, by BD (2 mentions)
28 protocols using anti mouse cd16 cd32 fc block
1
Comprehensive Murine Lung Cell Analysis
2
Quantifying Activated CD8+ T Cells via pSTAT5
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Red blood cell-depleted splenocytes from an IL-7 transgenic mouse were incubated with Fc-Block (anti-mouse CD16/CD32, BD) for 20 min on ice, followed by washing in flow cytometry buffer and staining with fluorophore-conjugated anti-CD8a and anti-CD44 antibodies. Stained cells were seeded in a 96-well plate (1 × 106 cells per well) and re-suspended in serially diluted IL-2-Fc variants. Stimulation was allowed for 10 min at 37 °C, followed by washing with flow cytometry buffer, fixation, permeabilization and intracellular staining with AF488-conjugated anti-pSTAT5 (clone 47/Stat5-pY694, BD). The levels of pSTAT5 in the CD8+ CD44high population were determined by flow cytometry. For flow cytometry of human PBMC, the following antibodies were used: Pacific Blue-conjugated anti-CD8 (clone RPA-T8, BD), APC-conjugated anti-CD4 (clone S3.5, Thermo Fisher), PE-conjugated anti-FoxP3 (clone 259D/C7, BD). Stimulation of PBMC was allowed for 10 min at 37 °C in the presence of 1 μg ml−1 (Treg stain) or 10 μg ml−1 (CD8+ T-cell stain) IL-2-Fc. Detection of intracellular pSTAT5 was performed as described above. Buffy coats from normal donors were obtained from the Australian Red Cross Blood Service. Informed consent was obtained from all subjects and approval for this study was obtained from the human research ethics committee of the St Vincent's Hospital (Sydney, Australia).
3
Quantifying Immune Cell Populations in BAL
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Cell suspension of BAL was centrifuged at 4 °C, 2,900 rpm for 10 min, and cell viability was determined by incubation with fixable viability dye (FVD) eFluor780 (eBiosciences) for 20 min. For subsequent flow cytometry (Gallios; Beckman Coulter), cells were fixed with 4% paraformaldehyde (PFA) and then incubated with Fc block (anti-mouse CD16/CD32; BD Biosciences) on ice for 20 min, followed by surface marker staining with anti-mouse Abs: F4/80 (PE; BD), CD11b (PE-CF594; BD), CD11c (PE-Cy7; eBiosciences), Ly6G (APC; BD), and Ly6C (V450; BD). The data were analyzed using Kaluza 1.2 (Beckman Coulter). The absolute numbers of different cell types were calculated based on the proportion of viable events analyzed by flow cytometry as related to the total number of viable cells per sample.
4
Quantifying Tumor-Infiltrating Immune Cells
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B16F10 tumours were dissected and carefully separated from skin tissue. Collected tumours were weighted and collected into serum-free RPMI 1640 (Thermo Fisher) containing 2 mg ml−1 collagenase D (Roche) plus 0.1 mg ml−1 DNAse I (grade I, Roche) and digested for 1 h at 37 °C. Tissue was then dispersed through 70 μm cell strainers (BD) to obtain single cell suspensions. The volume of cell suspension utilized for immunostaining was recorded for each sample (volume equivalent to ∼20 mg), followed by aliquoting into a 96-well plate and addition of 5 × 104 counting beads (FITC Calibrite, BD) per well. Samples were then incubated with Fc-Block (anti-mouse CD16/CD32, BD), surface antibody stain and fixable viability dye (eFluor780, eBioscience) in that order. This was followed by fixation/permeabilization (FoxP3 buffer set, eBioscience) and incubation with intracellular stain. Samples were then analysed by flow cytometry, as described above. Total numbers of immune subsets per gram of tumour were calculated utilizing the following formula: [(no. of acquired cells÷no. of acquired beads) × no. of added beads]÷[(volume stained÷total volume) × tumour weight in grams].
5
Tissue Sample Dissociation Protocol
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Tissue samples (tumors and TDLNs) were digested in an enzyme solution (dispase 0.2 µg/ml, collagenase A 0.2 µg/ml, DNase I 100 µg/ml, all Sigma-Aldrich/Merck, Darmstadt, Germany, in DMEMc), 1 ml per sample for 45 min at 500 rpm and 37°C. Cells were filtrated through 100 µm sterile strainers (Cell Trics, Partec, Sysmex Europe GmbH, Goerlitz, Germany), centrifuged at 300g +4°C for 5 min and the supernatant was discarded. The pellet was resuspended in 100 µl PBS containing Fc block (anti-mouse CD16/CD32 (BD, Franklin Lakes, US) and incubated for 15 min at +4°C.
6
In Vivo Labeling of Circulating T Cells
7
Ascites Volume and Immune Cell Analysis
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Mice were sacrificed on day 5 after last OV (monotherapy) or anti-PD-1 (combination therapy) injection, ascites was collected for volume measurement, and the cells were collected for flow cytometry, blocked with anti-mouse CD16/CD32 (Fc block, BD Bioscience) and stained with fluorescence-labeling antibodies at 4°C for 30 min. For intracellular staining, cell surface markers were stained, then cells were permeabilized with a FoxP3 fixation and permeabilization kit (eBioscience) for Ki-67 staining or with a Fixation/Permeabilization Kit (BD Bioscience) for IFN-γ staining. Cells used for IFN-γ staining were pretreated with Leukocyte Activation Cocktail containing Brefeldin A (BD Bioscience). In some analyses, fluorescence-minus-one was used to identify positive signals.
8
Lung Leukocyte Profiling in Mice
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In mouse models, BAL was performed by cannulation through the trachea and lavage with 1.5 ml sterile PBS. Cells were pelleted by centrifugation, resuspended in ACK buffer to lyse red cells, washed in PBS and resuspended in RPMI medium with 10% FBS. Cells were stained with Quik Diff (Reagena) for differential counting. For flow cytometry analysis, lung leukocytes were obtained from BAL and red cells were lysed with ACK buffer. BAL cells were stained with Live/Dead fixable dead cell stain (Life Technologies, Carlsbad, CA), incubated with anti-mouse CD16/CD32 (FC block; BD Biosciences) and subsequently with directly fluorochrome-conjugated monoclonal antibodies specific for CD3ε (clone 500 A; 2 1 µg/ml, BD Biosciences), CD69 (clone H1.2F3 1 µg/ml, BD Biosciences), CD4 (clone RM4-5 0.25 µg/ml; BD Biosciences), CD8a (clone 53–6.7; 0.5 µg/ml BD Biosciences) and NK1.1 (clone PK136;1 µg/ml, BD Biosciences). Data were acquired on an LSR II flow cytometer (BD Biosciences) and analysed using FlowJo software (version 10.0.6; Tree Star, Ashland, USA). Representative gating strategies used for analysis of cell surface staining are shown in Supplementary Fig. 8 .
9
Splenic Immune Profiling in Infected Mice
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The spleens of mice were collected at various time points post-infection, as detailed in figure legends. Splenic cells were passed through a 70 mm cell strainer (Fisher Scientific), centrifuged, and then treated with Ammonium-Chloride-Potassium (ACK) red blood cell lysing buffer (Gibco). Following washes, 4 x 106 total spleen cells were incubated with anti-mouse CD16/CD32 (Fc Block, BD Biosciences; Mississauga, ON, Canada) for 15 min on ice according to manufacturer’s instructions. Afterwards, surface staining with the following antibodies: anti-CD95PE, anti-CD86biotin, anti-CD19PE-Cy7, anti-GL-7AF647 and anti-CD184BV421 (all BD Biosciences) was performed for 30 min on ice. Following washing, cells were incubated with Brilliant Stain Buffer (BD Biosciences) and stained with StreptavidineBV605 (BD Biosciences) for 10 min on ice. Cells were then washed and immediately analyzed on a BD FACSaria Fusion. The data collected was analyzed on FlowJo version 10. All antibodies used are listed in S1 Table
10
Lineage Depletion of Bone Marrow Cells
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BM cells (1 × 107 cells/mL) were prepared in PBS containing 3% FCS, incubated on ice for 15 min with 2.5 μg anti-mouse CD16/CD32 (Fc Block, BD Biosciences) per 107 cells, and then incubated on ice for 15 min with Biotin Mouse Lineage Depletion Cocktail (BD Biosciences) consisting of biotinylated monoclonal anti-mouse CD3e, CD11b, CD45R/B220, Ly-6G, Ly-6C, and TER-119. Subsequently, the mixture was washed with 10 volumes of PBS containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid, and centrifuged. The resulting cell pellets were mixed with Streptavidin Particles Plus-DM (BD Biosciences), incubated at 6–12 °C for 30 min, and lineage-positive cells were removed using BD IMagnet (BD Biosciences) according to the manufacturer’s protocol. The depleted fractions were then used as lineage-negative BM cells, which have about 10-fold higher colony-forming activity than crude BM cells.
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