Leukocyte viability was determined by staining with Zombie UV Fixable Viability Dye (BioLegend, San Diego, CA). Cells were incubated for 5 min at 4 °C with anti-CD16/32 (Becton Dickinson; BD, San Jose, CA, 2.4G2), then stained with the following antimouse antibodies: CD45.1 (Thermo Fisher, A20), CD45.2 (Thermo Fisher, 104), CD11b (Thermo Fisher, M1/70), CD11c (BD, HL3), Ly6C (BioLegend, HK1.4), Ly6G (BD, 1A8), F4/80 (BD, T45-2342), B220 (Thermo Fisher, RA3-6B2), NKp46 (BD, 29A1.4), CD3 (Thermo Fisher, 17A2), MHCII (Thermo Fisher, M5/115.15.2), and Siglec-F (BD, E50-2440). Cells were fixed with stabilizing fixative (BD), and data were acquired on a BD Special Order Research Product LSRFortessa flow cytometer. Quality control checks with Cytometer Setup and Tracking beads (BD) and Ultra Rainbow Calibration Particles (Spherotech, Lake Forest, IL) were performed before each acquisition. Data were cleaned using FlowAI32 (link) and analyzed using Flowjo software (BD).
Anti cd16 32
Manufactured by BD
Sourced in United States, Japan
Anti-CD16/32 is a laboratory reagent used to block Fc receptors in flow cytometry and other immunoassay experiments. It binds to the CD16 and CD32 antigens, which are expressed on the surface of various immune cells, such as monocytes, macrophages, and natural killer cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (8 mentions)
Cytofix cytoperm, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Facscalibur, by BD (4 mentions)
Cytofix cytoperm kit, by BD (4 mentions)
Golgiplug, by BD (3 mentions)
Lsr 2, by FlowJo (3 mentions)
Anti iba1, by Fujifilm (3 mentions)
Collagenase a, by Merck Group (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Cd4 apc, by BD (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Brefeldin a, by BD (2 mentions)
Anti ifn γ apc, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Round bottomed 96 well plate, by Corning (2 mentions)
Perm wash buffer, by BD (2 mentions)
Anti cd8 percp, by BD (2 mentions)
Alexa fluor 594 anti vimentin clone epr3776, by Abcam (2 mentions)
Apc anti cd31 clone 390, by Thermo Fisher Scientific (2 mentions)
78 protocols using anti cd16 32
1
Flow Cytometric Immunophenotyping of Leukocytes
2
Murine Hepatic Mononuclear Cell Isolation
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Murine livers were passed through a 200-gage nylon mesh, and washed with cold PBS. The cell mixture was centrifuged at 50 × g for 2 min. The supernatant was then centrifuged at 800 × g for 10 min. For hepatic MNC isolation, the cell pellets were resuspended in 40% Percoll and centrifuged at 1250 × g for 15 min. Then the cell pellets were treated with lysis solution to remove erythrocytes and obtain hepatic MNCs. Next, cells were blocked with anti-CD16/32 (Becton Dickinson, San Diego, CA, USA) and labeled with anti-mouse CD4 antibodies (Becton Dickinson) before permeabilization with Cytoperm/Cytofix (Becton Dickinson) according to the manufacturer’s instructions. After permeabilization, the cells were incubated with labeled antibodies that were specific for FoxP3 (Becton Dickinson). Then, the cells were centrifuged, and the pellets were washed to remove unbound antibodies. After surface and intracellular labeling, MNCs were evaluated by flow cytometry (Calibrate; Becton Dickinson, Palo Alto, CA, USA) and the data were analyzed using FlowJo version 10 software (FlowJo, Ashland, OR, USA).
3
Cell Surface Marker Expression Analysis
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To measure the cell surface expression of CD47, HER2, EGFR, and CD20, cells were stained with conjugated primary antibodies (BD Biosciences) for 30 minutes at 4°C, washed, and resuspended in staining buffer. Macrophages were incubated with anti-CD16/32 (BD Biosciences) for 15 minutes at room temperature to block nonspecific binding and stained with CD14, CD68, CD80, CD163, CD206, PD-L1, and HLA-DR (BD Biosciences). Cells were acquired using a BD LSR X-20, and the data were analyzed using FlowJo software.
4
Detailed Immune Cell Phenotyping
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After homogenization of spleen tissue, immune cells were purified using 35% Percoll (GE Healthcare, La Chapelle-sur-Erdre, France) and red blood cells were lyzed. 106 leucocytes were incubated with anti-CD16/32 (BD Pharmingen, Rungis, France) to block non-specific binding and washed. Cells were then incubated 30 min with appropriate dilutions of anti-CD3-Pacific Blue, anti-CD8-APC-Cy7, anti-CD4-PE, anti-NK1.1-Percp-Cy5.5 and anti-CD19-APC antibodies, all purchased from BD Pharmingen. The staining of ST2 was assessed with a rat monoclonal anti-mouse ST2-FITC antibody (clone DJ8; MB Bioproducts). Cells were washed, fixed in PBS containing 2% FCS, 0.01 M sodium azide and 2% formaldehyde and analyzed on a FACS Aria II ® flow cytometer using BD FACS Diva software (BD Biosciences). Dead cells and doublet cells were excluded on the basis of forward and side scatter. The different immune cell types were identified and gated as follows: BL were CD19+; NK cells were NK1.1+/CD3−; T CD8+ lymphocytes were NK1.1−/CD3+/CD8+/CD4−; and T CD4+ lymphocytes were NK1.1−/CD3+/CD4+/CD8−. The gating strategy was previously described [5 (link), 12 (link)].
5
Flow Cytometry Analysis of Macrophages
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Flow cytometry analysis of nontargeting control and siGAPLINC-treated MDMs stained with CD11B FITC (Thermo Fisher), CD16 PE (Thermo Fisher), and CD14 FITC (Biolegend).
Blood was collected immediately postmortem by cardiac puncture, and single-cell suspensions prepared from the spleen of WT and Gaplinc-KO mice were depleted of red blood cells (RBCs) prior to staining. Fragment, crystallizable (Fc) receptors were blocked (anti-CD16/32, BD Pharmingen) prior to staining with LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher), anti–CD11b-AlexaFluor 700, anti–LY6G-BV421, anti–LY6C-AlexaFluor 488, anti–CD19 PerCP-Cy5.5, anti–CSF-1R APC (BioLegend), anti–CD3-PE (BD), anti–SiglecF BV650 (BD OptiBuild), and anti–F4/80 PE-eFluor 610 (eBioScience).
Flow cytometry analysis of peritoneal fluid isolated from WT and Gaplinc-KO mice (24 (link)) challenged with LPS i.p. for 18 h was performed; cells were stained with anti–CD11b-AlexaFluor 700 and anti–F4/80 PE-eFluor 610 (eBioScience).
Flow cytometry analysis of BMDMs harvested from WT and Gaplinc-KO mice was performed; cells were stained with anti–CD11b-AlexaFluor 700 and anti–F4/80 PE-eFluor 610 (eBioScience).
Data acquisition was performed using Attune NxT (Thermo Fisher). Analysis was performed using FlowJo analysis software (BD Biosciences).
Blood was collected immediately postmortem by cardiac puncture, and single-cell suspensions prepared from the spleen of WT and Gaplinc-KO mice were depleted of red blood cells (RBCs) prior to staining. Fragment, crystallizable (Fc) receptors were blocked (anti-CD16/32, BD Pharmingen) prior to staining with LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher), anti–CD11b-AlexaFluor 700, anti–LY6G-BV421, anti–LY6C-AlexaFluor 488, anti–CD19 PerCP-Cy5.5, anti–CSF-1R APC (BioLegend), anti–CD3-PE (BD), anti–SiglecF BV650 (BD OptiBuild), and anti–F4/80 PE-eFluor 610 (eBioScience).
Flow cytometry analysis of peritoneal fluid isolated from WT and Gaplinc-KO mice (24 (link)) challenged with LPS i.p. for 18 h was performed; cells were stained with anti–CD11b-AlexaFluor 700 and anti–F4/80 PE-eFluor 610 (eBioScience).
Flow cytometry analysis of BMDMs harvested from WT and Gaplinc-KO mice was performed; cells were stained with anti–CD11b-AlexaFluor 700 and anti–F4/80 PE-eFluor 610 (eBioScience).
Data acquisition was performed using Attune NxT (Thermo Fisher). Analysis was performed using FlowJo analysis software (BD Biosciences).
6
Measuring iNKT Cell Response by Flow Cytometry
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Cell population and cytokine secretion of iNKT cells were measured by flow cytometry. Before staining cells, with a previously defined optimal dilution of monoclonal antibodies (Abs), the cells were pre-incubated with anti-CD16/32 (clone 93) to block non-specific FcRγ binding. The following Abs were used in this study: anti-CD3, anti-CD4, anti-CD8, and anti-CD49b (Biolegend, San Diego, CA, USA). For intracellular staining, BALF cells were incubated with brefeldin A (10 μg/ml) (BD Biosciences, San Diego, CA, USA) at 37°C for 1 hour then incubated with anti-CD16/32 Abs, followed by staining with PerCP/Cy5.5-conjugated CD3 and PE-conjugated PBS57 loaded CD1d tetramer (originally produced by the NIH tetramer facility, and supplied through Dr. David Serreze), permeabilized with Cytofix/Cytoperm reagent (BD Biosciences), and stained with Alexa Fluor® 488-conjugated anti-IFN-γ (clone XMG1.2), Alexa Fluor® 488-conjugated anti-IL-4 (clone 11B11), or rat IgG1 isotype control (clone R3-34) (BD Biosciences). Stained cells were assessed on a FACSCalibur (BD Biosciences) using FlowJo softwares (Tree Star, Inc., Ashland, OR, USA).
7
Lung Cell Isolation and Analysis
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Whole lungs were perfused with saline and minced using a tissue chopper (Campden Instruments, Lafayette, IN, http://campdeninstruments.com ). Lung tissues were digested in Dulbecco’s modified Eagle’s medium‐F12 media (11330‐057; Thermo Fisher Scientific Life Sciences, Waltham, MA, http://www.thermofisher.com ) containing 25 mg/ml collagenase IA (10103578001; Roche, NSW, Australia, http://www.roche.com ), 2.5 mg/ml DNase I (AMPD1; Sigma‐Aldrich, St. Louis, MO, http://www.sigmaaldrich.com ), and 10% (volume per volume) heat‐inactivated fetal bovine serum (16110‐082; Thermo Fisher) for 15 minutes at 37°C. Lung lysates were passed through a 70‐μM cell strainer and red blood cells were lysed. Fc receptors were blocked with anti‐CD16/32 (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com ) before staining for CD45, CD4, CD11b, F4/80, Ly6C, and CD11c. Data were acquired using a BD LSR II analyzer (BD, Franklin Lakes, NJ, http://www.bd.com ). Representative gating strategies are shown in supplemental online Figure 1.
8
Evaluating CD4+ T Cell Proliferation
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15 days post immunization, single cell suspensions were generated by isolating the LNs of the immunized mice. Utilizing the CellTrace™ CFSE (5(6)‐carboxyfluorescein N‐hydroxysuccinimidyl ester) Cell Proliferation kit (Life Technologies, Carlsbad, CA), CD4+ T cell proliferation against antigens was determined. Briefly, isolated 20 × 106 LN cells were incubated for 5 minutes at room temperature with 1 μmol/L CFSE. After incubation, cells were washed with RPMI media twice, then incubated in a 96‐well‐round bottom plate at 1 × 106 cells per well with specified antigen for 96 h. Post incubation, cells were washed with staining FACS buffer two times, then the Fc receptors were blocked with anti‐CD16/32 (BD Biosciences, Franklin Lakes, NJ,) for 15 min at 4°C before staining with mAbs for 30 min at 4°C. Cells were stained utilizing the following monoclonal antibodies: CD3e‐Pacific Blue, CD45‐PE‐Cy7, and CD4‐PE‐Texas Red. Cells were analyzed with a LSRII flow cytometer (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR, USA).
9
Phenotyping Lymphocyte Populations by Flow Cytometry
10
CD8+ T Cell Intracellular Cytokine Assay
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One million splenocytes were added to each well of a 96-well round-bottomed plate (Costar, Corning, NY), pulsed with 2 μg/mL P18-I10 peptide and kept at 37°C and 5% CO2 for 60 min, followed by the addition of GolgiStop (Becton Dickinson) containing monensin. After 5 h of incubation, the reaction was terminated by transferring the plate to 4°C. The cells were washed with wash buffer (PBS, 2% fetal calf serum, and 0.01% azide) and blocked with anti-CD16/32 (BD Biosciences) at 4°C for 30 min. All subsequent antibody stains were performed using the same conditions. Cells were then washed and stained with anti-CD8-PerCP (BD Biosciences) and anti-CD107a-fluorescein isothiocyanate (FITC), washed again, and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences). Perm-wash buffer (BD Biosciences) was used to wash cells before staining with anti-IFN-γ-APC and anti-TNF-α-PE (BD Biosciences). Cells were fixed with CellFIX (Becton Dickinson) and stored at 4°C until analysis. All chromogen-labeled cells were analyzed in a Becton Dickinson FACScalibur, using the CellQuest software (Becton Dickinson) for acquisition and the FlowJo software (Tree Star, Ashland, OR) for analysis.
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