The spleen and lymph nodes were homogenized using 70 μm cell strainers (BD Biosciences San Jose, CA, USA) to produce single cell suspensions. Red blood cells from spleens were lysed with Pharmlyse (BD Biosciences) and then washed with D-PBS (Sigma, St. Louis, MO, USA) + 1% FBS buffer. Nonspecific antigen binding was blocked using 5 μg/ml CD16/32 (clone 2.4-G2, BD Bioscience). One million cells were stained for 30 min on ice with fluorochrome-conjugated FITC-Ly6C (BD Biosciences,), PE-CD11b (1:400) and PE-Gr-1 (1:200) to gate out monocytic cells and FITC-CD4 (1:300) and PerCP-eFluor710-CD8 (1:600) (eBioscience, San Diego, CA, USA). Samples were washed twice in buffer and analyzed on a FACSCalibur (BD Biosciences) equipped with a single 488-nm argon laser. Data analysis was performed using Cellquest Pro software (BD Biosciences).
Cd16 32 clone 2.4 g2
Manufactured by BD
Sourced in United States
CD16/32 (clone 2.4-G2) is a laboratory reagent used in flow cytometry applications. It binds to the CD16 and CD32 receptors expressed on various immune cells, including natural killer cells, monocytes, and macrophages. This reagent can be used to identify and characterize these cell types in research or diagnostic settings.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (1 mentions)
Pharm lyse, by BD (1 mentions)
Cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Cd11b pe, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Pe gr1, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Live dead near ir stain, by Thermo Fisher Scientific (1 mentions)
Anti ly6c apc clone al 21, by BD (1 mentions)
Anti mhcii bv421 clone m5 114.15.2, by BD (1 mentions)
Anti cd11b pe cy7 clone m1 70, by BD (1 mentions)
Chamber slide, by Corning (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Clone 30 f11, by BD (1 mentions)
Cd31 clone mec 13, by BD (1 mentions)
Dispase, by BD (1 mentions)
Dnase, by Serva Electrophoresis (1 mentions)
5 protocols using cd16 32 clone 2.4 g2
1
Multiparametric Flow Cytometric Analysis of Immune Cells
2
Multicolor Flow Cytometry of Footpad Cells
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Single cell suspensions of footpad cells were filtered over a 70-µm nylon filter mesh and incubated in saturating doses of purified anti-mouse Fc receptor (CD16/32, clone 2.4G2, BD Bioscience) in 1 ml PBS + 5% FCS for 10 min on ice to prevent antibody binding to Fc receptor. The isolated cells were stained on ice with various fluorescent monoclonal antibodies (mAbs) combinations in PBS + 5% FCS for 30 min. Anti-Ly6G-FITC (clone 1A8, BioLegend), anti-CD18-PE (clone M18/2, eBioscience), anti-CD11b-PECy7 (clone M1/70, BD Biosciences), anti-Ly6C-APC (clone AL-21, BD Biosciences), anti-CD62L-APCCy7 (clone MEL14, eBioscience), anti-MHCII-BV421 (clone M5/114.15.2, BD Biosciences), and anti-CD45-BV510 (clone 30-F11, BD Biosciences). A live/dead near-IR stain (ThermoFischer Scientific) was performed in PBS only. Unbound mAbs were discarded by washing the samples with PBS + 5% FCS and centrifugation (1,400 rpm, 7 min, 4°C). Analyses were performed using a FACS Canto II flow cytometer (BD Biosciences) and data were processed using FlowJo software (Tree Star Inc.). Cells were gated according to size and scatter to eliminate debris from analysis.
3
Isolation and Culture of Alveolar Epithelial Cells
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Lung homogenates were obtained by instillation of dispase (BD Biosciences) through the trachea into HBSS (Gibco) perfused lungs, followed by incubation (in dispase) for 40 min as previously described61 (link). After removal of the trachea and proximal bronchial tree, the lungs were homogenized (GentleMACS, MACS Miltenyi Biotech) in DMEM/2.5% HEPES with 0.01% DNase (Serva) and filtered through 100 and 40 μm nylon filters. Cell suspensions were incubated with biotinylated rat anti-mouse CD45 (Clone 30-F11, BD, Cat No 553078), CD16/32 (clone 2.4G2, BD, Cat No 553143) and CD31 (clone MEC13.3, BD, Cat No 553371) mAbs for 30 min at 37 °C followed by incubation with biotin-binding magnetic beads and magnetic separation to deplete leukocytes and endothelial cells prior to further culture. AEC suspensions with a purity ≥90% as determined by FACS were seeded at a density of 120–150,000 cells/cm2 in 4-μm–pore size transwells (Corning Inc.), in 24-well plates at a density of 250,000 cells/cm2, or in chamber slides (Corning Inc.), and cultured in DMEM enriched with HEPES, L-Glutamine, FCS, and pen/strep. The list of antibodies in Excel format including clone, company name, Cat No is provided as Supplementary Data 1 .
4
Flow Cytometric Analysis of Splenic Immune Cells
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For flow cytometric analysis of splenic mononuclear cells, up to 2 × 106 cells were added to a 96-well plate. The isolated cell suspension was washed with PBS, incubated with Fcγ receptor blocker CD16/32 (clone 2.4G2; BD Pharmingen), and stained with specific monoclonal antibodies at the indicated concentration in PBS solution containing 1% FBS. The following antibodies were used: fluorescently-conjugated antibodies (BD Pharmingen) against mouse CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD19 (1D3), CD25 (3C7), Foxp3 (R16-715), CD69 (H1.2F3), PD-1 (J43), Tim-3 (5D12), and CD28 (37.51). Surface antibody staining was performed at 4°C for 30 min in the dark. For Tregs staining, the cells stained with surface molecules were fixed, permeabilized, and stained for intracellular Foxp3 using the Transcription Factor Buffer kit according to the manufacturer’s instructions (BD Pharmingen). After staining, the cells were washed three times with PBS, and fixed with 0.5% paraformaldehyde at 4°C. The stained cells were analyzed on a FACS Aria III flow cytometer (BD Biosciences). The acquired data was analyzed with FlowJo software (Tree Star).
5
Multiparametric Flow Cytometry Analysis
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Cells were stained with respective anti-mouse monoclonal antibodies (Supplementary Table S1 ) for 30 min at 4°C, washed, and resuspended in sterile FACS buffer, containing 0.1% bovine serum albumin in phosphate buffered saline (PBS; Lonza, Basel, Switzerland). A 15 min long Fcγ receptor blocking step (unlabelled CD16/32, clone 2.4G2; BD Biosciences, Franklin Lakes, New Jersey, USA) preceded all stainings. Data were acquired on a FACSCantoII (BD Biosciences) and analyzed using FlowJo software V10 (FlowJo LLC, Ashland, Oregon, USA). Cells were sorted by FACSAria Fusion (Becton-Dickinson) at Research Facility Cell Sorting of Hannover Medical School. Apoptosis was assessed with FITC Annexin V Apoptosis Detection Kit (BD Biosciences).
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