After 6 days of culture, the cells were harvested by cell scraper, and a single cell suspension was prepared in phosphate-buffered saline (PBS) containing 10% of FBS on ice. Fc blocking was performed using rat anti-mouse CD16/CD32 (553141, BD Pharmingen, Franklin Lakes, NJ, USA) for 20 min on ice [23 (link)]. After that, cell surface staining was performed by adding anti-mouse F4/80 (Brilliant Violet 605, 123133, Biolegend, San Diego, CA, USA) and incubated for 30 min on ice in the dark. The mouse lymph node cells were used as a positive control. After washing twice with PBS, the cells were then resuspended in ice-cold PBS with 10% FBS for the flow cytometric analysis using a BD LSRFortessa cell analyzer (BD Biosciences, San Jose, CA, USA). The mouse bone marrow cells and lymph node cells were kept in cell freeze media (Bambanker, NIPPON Genetics, Bunkyo-ku, Tokyo, Japan) and were stored at −80 °C before experiments.
Rat anti mouse cd16 cd32
Manufactured by BD
Sourced in United States
The Rat anti-mouse CD16/CD32 is a laboratory reagent used for the identification and characterization of mouse CD16 and CD32 receptor molecules. It is a monoclonal antibody that can be used in various immunological techniques such as flow cytometry and cell sorting to detect the presence of these receptors on the surface of mouse cells.
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Lab products found in correlation
Lsrfortessa, by BD (4 mentions)
Fixation permeabilization kit, by BD (3 mentions)
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Goat anti rat f ab 2, by Jackson ImmunoResearch (2 mentions)
Rat serum, by GeneTex (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Facsverse, by BD (2 mentions)
Anti cd8 53 6.7, by BioLegend (2 mentions)
Anti cd4 rm4 5, by BioLegend (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Cd11b, by BioLegend (2 mentions)
Facs lysing solution, by BD (2 mentions)
Pe rat anti mouse cd71, by BD (2 mentions)
Facsariaii high speed cell sorter, by BD (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Methylbutane, by Merck Group (2 mentions)
Polyclonal guinea pig anti insulin antibody, by Agilent Technologies (2 mentions)
Tcs sp5 confocal microscopy, by Leica (2 mentions)
Alexafluor 647 conjuaged goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
48 protocols using rat anti mouse cd16 cd32
1
Murine Macrophage Immunophenotyping by Flow Cytometry
2
Isolation and Stimulation of Tumor-Associated Macrophages
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TAMs were isolated from Panc02 PDAC tumors based on FACS sorting of cells stained with CD11b and F4/80. TAMs were stimulated for Fc gamma ligation or LPS or α4β1 integrin stimulation. For Fc gamma ligation, cells were incubated with purified rat antimouse CD16/CD32 (BD Pharmingen) at 1.0 μg/106 cells for 10 minutes 4°C and then incubated with goat anti-rat F(ab’)2 (25 μg/mL; Jackson ImmunoResearch) for 10 minutes, followed by lysate preparation. For TLR4 signaling, TAMs were stimulated with 1 μg/mL of LPS for 10 minutes, followed by lysate preparation. For α4β1 integrin studies, nontissue culture petri dishes were coated with 10 μg/mL of H296 (ligand for α4β1 integrin) in PBS for 1 hour, followed by washing with PBS and plating of cells for 10 minutes and lysate preparation.
3
Multicolor Flow Cytometry of Mouse Splenocytes
4
Single-Cell Sorting of Tumor-Infiltrating CD45+ Cells
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For sorting of CD45+ cells for single-cell RNA sequencing, single-cell suspensions of tumors were blocked with purified rat anti-mouse CD16/CD32 (BD Pharmingen, dilution 1:100) for 10 min at room temperature then stained with Live/Dead dye (Zombie Aqua, Biolegend) and anti-mouse CD45 (BV605 or APC-Cy7, Biolegend) for 25 min on ice. Live CD45+ cells were isolated for scRNA-seq using an Astrios (Beckman Coulter) sorter and resuspended in PBS with 0.04% BSA at a concentration of 1000 cells/µL for single-cell RNA sequencing.
5
Multiparametric Flow Cytometry Analysis
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For flow cytometry, 1 × 106 cells were used per sample unless otherwise indicated. A mix of 0.05 mg/mL purified rat anti-mouse CD16/CD32 (BD Pharmingen), rat serum (GeneTex, Irvine, CA) and hamster serum (Jackson Immunoresearch, West Grove, PA) was used to block Fc receptors on tumor infiltrating leukocytes for 20 min at 4 °C. This step was omitted for tumor cell lines and splenocytes. The cells were then washed twice and 100 μL of LIVE/DEAD® Fixable Yellow Dead Cell Stain or LIVE/DEAD® Fixable Blue Dead Cell Stain Kit, for UV excitation (Life technologies / Thermo Fisher) diluted 1:1000 in PBS was added. After incubation for 30 min at 4 °C, washed cells were stained with antibodies (or isotype controls) listed in Additional file 1 : Table S1. After incubation for 1 h at 4 °C, cells were washed and resuspended in 200–400 μL FACS buffer for flow cytometric sorting or analysis, respectively. For intracellular cytokine staining, cells were incubated in 100 μL fixation permeabilization solution (BD Biosciences, San Jose, CA) for 20 min at 4 °C, followed by two washing steps using BD Perm/Wash buffer. Cells were then incubated for 1 h at 4 °C with 100 μL BD Perm/Wash buffer containing antibodies or isotype controls, respectively, diluted 1:100. Finally, cells were resuspended in 200–400 μL FACS buffer for analysis.
6
Tumor Cell Phenotyping by Flow Cytometry
7
Aortic Endothelial Cell Immunofluorescence
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Aortas from euthanized mice were isolated after perfusion with saline followed by 4% paraformaldehyde. Aortas were then longitudinally cut and pinned to a gelatin-coated plate and fixed in 4% paraformaldehyde overnight at 4 °C. The tissue was permeabilized and blocked with 0.3% Triton X-100, 10% goat serum, 5% BSA, and FcBlock (1:100; Rat Anti-Mouse CD16/CD32; BD Bioscience) in PBS for 1 h. Aortas were incubated overnight at room temperature hamster anti-cd31 (1:200; MAB1398Z; Millipore) in blocking buffer. After abundant washes with PBS, samples were incubated overnight with AlexaFluor647-conjugated goat anti-hamster secondary antibody (1:500, 127-605-160, Jackson Inmuno Reasearch) and DAPI (1:10,000). Aortas were mounted in Citifluor AF4 mounting medium (Aname). Images were acquired with a Nikon A1R confocal microscope fitted with a 20x air objective or a 40x oil immersion objective and using Nikon NIS-Elements software (1024 × 1024 pixels, 8bits).
8
Lung and BALF Immune Cell Profiling
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Cells from BALF or lung cell suspensions were washed with FACS buffer (0.1% FBS and 0.1% NaN3 in PBS) and blocked with purified rat anti–mouse CD16/CD32 (553141, BD Pharmingen) according to the vendor’s instruction. Neutrophils in BALF were stained with APC-conjugated anti-CD45 Ab and BV510-conjugated anti-Ly6G Ab; after washing, cells were resuspended in FACS buffer, and the CD45+Ly6G+ neutrophils were detected by flow cytometry. Lung cell suspensions were stained with BUV805-conjugated anti-CD45 Ab, BV650-conjugated anti-Ly6G Ab, BV480-conjugated anti-CD11c Ab, BUV395-conjugated anti-CD11b Ab, BV605-conjugated anti-CD64 Ab, PE-Cy5-conjugated anti-CD24 Ab, and BUV496-conjugated anti-IA/IE Ab, and cell populations were determined by flow cytometry (BD FACSVerse).
9
Intracellular Cytokine Staining of T Cells
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To analyze cytokine-producing T cell populations, the lung lymphocytes were resuspended in IMDM supplemented with 10% heat-inactivated fetal bovine serum (10% IMDM) and stimulated with 10 μg of synthetic cryptic-epitope peptide (LSMPFHNIHPLTIGECPKYVKSVR) in 10% IMDM containing 10 ng of recombinant human IL-2 (BioLegend, San Diego, CA, USA) and Brefeldin A (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 5 h in dark. After stimulation, the cells were incubated with rat anti-mouse CD16/CD32 (BD Biosciences) blocking antibody for 10 min at room temperature, and then, the lung cells were stained with anti-CD4 (RM4-5; BioLegend) and anti-CD8 (53-6.7; BioLegend) antibodies for 30 min at 4°C in the dark. For intracellular cytokine staining, the stained cells were fixed with fluorescence-activated cell sorting lysing solution (BD Biosciences) for 20 min at room temperature, and permeabilized with fluorescence-activated cell sorting buffer (0.5% fetal bovine serum, 0.09% NaN3 in PBS) containing 0.5% saponin (MilliporeSigma) for 15 min at room temperature. Then, those cells were stained with anti-IFN-γ (XMG 1.2; BioLegend) for 30 min at room temperature in the dark. The stained cells were analyzed by LSR Fortessa (BD Biosciences), and all flow cytometry data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).
10
Immune Cell Isolation and Sorting
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Collected cell suspension were blocked with purified rat anti-mouse CD16/CD32 (BD Pharmingen, dilution 1:100) for 10 min, then stained with fluorophore conjugated antibodies. Antibodies used in this study are anti-mouse CD45-APCCy7, Ter119-APCCy7 (Biolengend), DAPI (eBiosciences) stain was used to exclude dead cells. For cell sorting, single cells were gated using doublet-discrimination parameters and cells were collected in FACS buffer (1 x HBSS, 2% FBS, 1 mM EDTA). Cell viability was assessed with trypan blue and only samples with >85% viability were processed for further sequencing.
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