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Anti cd16 32 clone 93

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Anti-CD16/32 (clone 93) is a monoclonal antibody that binds to the mouse CD16 and CD32 receptors. CD16 and CD32 are Fc receptors that play a role in the immune response.

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Lab products found in correlation

Dnase 1, by Merck Group (7 mentions) Live dead fixable yellow dead cell stain kit, by Thermo Fisher Scientific (6 mentions) Collagenase a, by Roche (5 mentions) Siglecf e50 2440, by BD (3 mentions) Lsrfortessa, by BD (2 mentions) Facsaria 2, by BD (2 mentions) Fortessa x20, by BD (2 mentions) Collagenase type 4, by Roche (2 mentions) Dnase type 1, by Roche (2 mentions) Rbc lysis buffer, by Thermo Fisher Scientific (2 mentions) Flavoperidol, by Merck Group (2 mentions) Zombie aqua viability dye, by BioLegend (2 mentions) Facsaria 2 flow cytometer, by BD (2 mentions) Collagenase, by Roche (2 mentions) Cd11b m1 70, by Thermo Fisher Scientific (2 mentions) Live dead aqua, by Thermo Fisher Scientific (2 mentions) Facsdiva software, by BD (2 mentions) Facsverse, by BD (2 mentions) Liberase, by Roche (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

Anti-CD16/32 (115 citations) CD16/32 (68 citations) Anti-mouse CD16/32 (clone 93, (17 citations) Anti-mouse CD16/32 antibody (clone 93, (12 citations) Anti-CD16/32 (93, (8 citations) Anti-CD16/32 antibody (clone 93, (8 citations) CD16/32 (93, (6 citations) CD16/32 (clone 93) (5 citations) Rat anti-mouse CD16/32 (clone 93; (4 citations) TruStain fcX anti-mouse CD16/32; clone 93 (4 citations)

30 protocols using anti cd16 32 clone 93

1

Multiparameter Flow Cytometry of Peritoneal Cells

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Red blood cells were lysed from peritoneal lavages, and equal numbers of cells were blocked with 0.025 μg anti-CD16/32 (clone 93; BioLegend, San Diego, CA, USA) and then stained with a combination of antibodies shown in Table 1. Fluorescence minus 1 and negative controls were used to confirm gating strategies. Just prior to analysis, DAPI and 123count eBeads allowing absolute numbers of cells to be determined (Thermo Fisher Scientific) were added to samples. Samples were acquired using an LSRFortessa with FACSDiva software (BD Biosciences) and analyzed with FlowJo v.9 software (FlowJo, Ashland, OR, USA). Analysis was performed on single live cells determined using forward scatter height vs. area and negativity for live or dead (DAPI).
Forster R., Sarginson A., Velichkova A., Hogg C., Dorning A., Horne A.W., Saunders P.T, & Greaves E. (2019). Macrophage-derived insulin-like growth factor-1 is a key neurotrophic and nerve-sensitizing factor in pain associated with endometriosis. The FASEB Journal, 33(10), 11210-11222.
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2

Quantifying Macrophage iNOS Expression

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Cells were incubated with anti-CD16/32 (clone 93, Biolegend), surface stained for F4/80 (clone BM8, eBioscience) and CD11b (clone M1/70; BD PharMingen) together with LIVE/DEAD dye (Invitrogen), followed by intracellular iNOS stain with anti-NOS2 (clone C-11; Santa Cruz Biotechnology) and FITC-conjugated IgG (Clone A85-1; BD PharMingen) using the BD cytofix/cytoperm Kit (BD Biosciences). Cells were acquired on Canto II flow cytometer (BD Biosciences), and data were analyzed with FlowJo v.9.5.2 software (Tree Star).
Lampropoulou V., Sergushichev A., Bambouskova M., Nair S., Vincent E.E., Loginicheva E., Cervantes-Barragan L., Ma X., Huang S.C., Griss T., Weinheimer C.J., Khader S., Randolph G.J., Pearce E.J., Jones R.G., Diwan A., Diamond M.S, & Artyomov M.N. (2016). Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage Metabolic Remodeling and Regulation of Inflammation. Cell metabolism, 24(1), 158-166.
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3

Multiparameter Flow Cytometry of Th Cell Subsets

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Single cell suspensions were obtained from spleen, PPs, and MLNs. Cells were stained with Fixable Viability Dye eFluor 506 (eBioscience, Vienna, Austria) for exclusion of dead cells. A-specific binding to Fc receptors was blocked by incubating the cells with anti-CD16/32 (clone 93, Biolegend, Uithoorn, the Netherlands) for 15 min on ice. For extracellular staining, cells were incubated with the desired mixture of antibodies for 30 min on ice. After washing, cells were fixed with FACS lysing solution (BD Biosciences, Breda, the Netherlands). For intracellular staining, fixed cells were permeabilized with PERM (eBioscience, Vienna, Austria) and subsequently stained with the desired antibodies for 30 min on ice. For identification of the different Th cell subsets, cells were stained with antibodies against: CD3e (clone 17A2), CD4 (clone GK1.5), T-bet (clone 4B10), RORγt (clone B2D), Gata-3 (clone TWAJ), CD25 (clone PC61), and Foxp3 (clone FJK-16S). Appropriate isotype controls were used to determine specificity of the staining. Samples were acquired with the FACSVerse (BD Biosciences, Breda) and analyzed with FlowJo software (FlowJo, LLC, Oregon, USA).
Fransen F., van Beek A.A., Borghuis T., Aidy S.E., Hugenholtz F., van der Gaast – de Jongh C., Savelkoul H.F., De Jonge M.I., Boekschoten M.V., Smidt H., Faas M.M, & de Vos P. (2017). Aged Gut Microbiota Contributes to Systemical Inflammaging after Transfer to Germ-Free Mice. Frontiers in Immunology, 8, 1385.
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4

Isolation and Flow Cytometric Analysis of Lung Immune Cells

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The lungs were removed, and single cells were isolated by enzymatic digestion with 1 mg/ml collagenase A (Roche, Indianapolis, IN) and 20 U/ml DNaseI (Sigma, St. Louis, MO) in RPMI 1640 containing 10% FCS. Tissues were further dispersed through an 18-gauge needle (10-ml syringe), RBCs were lysed and samples were filtered through 100-μm nylon mesh twice. Cells were resuspended in PBS and live cells were identified using LIVE/DEAD Fixable Yellow Dead Cell Stain kit (Thermo Fisher Scientific, Waltham, MA), then washed and resuspended in PBS with 1% FCS and Fc receptors were blocked with purified anti-CD16/ 32 (clone 93; BioLegend, San Diego, CA). Surface markers were identified using Abs (clones) against the following antigens, all from BioLegend: anti-Gr-1 (RB6-8C5), B220 (RA3-6B2), CD3 (145-2C11), Ter119 (Ter-119), CD11b (M1/70), CD25 (PC61), CD45 (30-F11), CD127 (SB/199), ST2 (DIH9), c-KIT (2B8) and CD90 (30-H12). SiglecF (E50-2440) was purchased from BD Biosciences (San Jose, CA). For innate lymphoid cell staining, lineage markers were anti-CD3, CD11b, B220, Gr-1, and TER119. ILC2: Lin−CD45+CD25+CD90+ST2+ c-Kit +CD127+. Eosinophils: SSChCD45+11b+SinglecF+. Mast cells: CD45+CD11b+cKIT+FcεR1+. Data was collected in NovoCyte flow cytometer (ACEA Bioscience, Inc. San Diego, California). Data analysis was performed using FlowJo software (Tree Star, Oregon).
Fonseca W., Rasky A.J., Ptaschinski C., Morris S.H., Best S.K., Phillips M., Malinczak C.A, & Lukacs N.W. (2019). Group 2 innate lymphoid cells (ILC2) are regulated by stem cell factor during chronic asthmatic disease. Mucosal immunology, 12(2), 445-456.
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5

FACS Analysis of Single Cell Suspensions

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Single cell suspensions were centrifuged at 400 g for 5 min, resuspended in 200 μL staining buffer (1X PBS, 1% BAS, 2 mM EDTA), placed in 5ml round-bottom tubes, and immunolabeled for FACS analysis. Before immunostaining, cell suspensions were pre-incubated with diluted (1:50) purified anti-CD16/32 (clone: 93, Biolegend, 101302) for 10 min on ice to block non-specific binding to Fcreceptors. Next, antibodies were added and incubated for 40 min on ice. Where appropriate, cells were further incubated with streptavidin conjugates for 20 min. All antibodies used can be found in key resources table. All FACS analyses were carried out on LSR Fortessa or Fortessa X-20 (BD Biosciences). Cell sorting was performed on FACS Aria II (BD Biosciences) sorter using 85 μm nozzle. All data were analyzed using FlowJo10 software (Tree Star).
Wu J., Krchma K., Lee H.J., Prabhakar S., Wang X., Zhao H., Xing X., Seong R.H., Fremont D.H., Artyomov M.N., Wang T, & Choi K. (2020). Requisite Chromatin Remodeling for Myeloid and Erythroid Lineage Differentiation from Erythromyeloid Progenitors. Cell reports, 33(7), 108395.
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6

Isolation and Characterization of Lung Mononuclear Phagocytes

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Mice were euthanized with carbon dioxide. Lungs were perfused with PBS trough the right ventricle until no blood was visible in the tissue. Lungs were removed, minced and placed in a digestion buffer containing collagenase type 4 (1.6 mg/ml; Roche), DNase type 1 (50 units/ml; Roche) and Flavoperidol (1 μM, Sigma-Aldrich) in RPMI 1640 medium. Lungs were incubated at 37 °C for 15 min while on a rotator. Red blood cells were lysed using RBC lysis buffer (eBioscience) at 4 °C for a 5 min incubation. The obtained cell suspension was filtered using a 70-micron filter, washed and cells were resuspended in PBS. Cells were then incubated with anti-Fc blocking mAb (anti CD16/32, clone 93, BioLegend) and viability dye Zombie Aqua (BioLegend) for 10 min. After this, cells where stained with an antibody cocktail containing APC Cy7-conjugated CD45, PE-conjugated F4/80, BV605-conjugated CD11c, v450-conjugated CD11b, PE Cy7-conjugated CD64, AF700-conjufgated Ly6c, FITC-conjugated Ly6g, AF647-conjugated SiglecF mAb for 30 min. All labeled cells were passed through a FACS Aria II flow cytometer (BD Biosciences) and after proper gating 4 subsets of lung MPs were sorted29 (link). Data were analyzed with FlowJo 8.3.3 software (TreeStar Inc.). The antibodies used for FACS are listed in Supplementary Table 1.
Sajti E., Link V.M., Ouyang Z., Spann N.J., Westin E., Romanoski C.E., Fonseca G.J., Prince L.S, & Glass C.K. (2020). Transcriptomic and epigenetic mechanisms underlying myeloid diversity in the lung. Nature immunology, 21(2), 221-231.
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7

FACS Analysis of Single Cell Suspensions

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Single cell suspensions were centrifuged at 400 g for 5 min, resuspended in 200 μL staining buffer (1X PBS, 1% BAS, 2 mM EDTA), placed in 5ml round-bottom tubes, and immunolabeled for FACS analysis. Before immunostaining, cell suspensions were pre-incubated with diluted (1:50) purified anti-CD16/32 (clone: 93, Biolegend, 101302) for 10 min on ice to block non-specific binding to Fcreceptors. Next, antibodies were added and incubated for 40 min on ice. Where appropriate, cells were further incubated with streptavidin conjugates for 20 min. All antibodies used can be found in key resources table. All FACS analyses were carried out on LSR Fortessa or Fortessa X-20 (BD Biosciences). Cell sorting was performed on FACS Aria II (BD Biosciences) sorter using 85 μm nozzle. All data were analyzed using FlowJo10 software (Tree Star).
Wu J., Krchma K., Lee H.J., Prabhakar S., Wang X., Zhao H., Xing X., Seong R.H., Fremont D.H., Artyomov M.N., Wang T, & Choi K. (2020). Requisite Chromatin Remodeling for Myeloid and Erythroid Lineage Differentiation from Erythromyeloid Progenitors. Cell reports, 33(7), 108395.
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8

Isolation and Characterization of Lung Immune Cells

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The lungs were removed, and single cells were isolated by enzymatic digestion with 2.5 mg/ml LiberaseTM (Roche) and 20 U/ml DNaseI (Sigma, St. Louis, MO) in RPMI 1640 for 45 min at 37 °C or 1 mg/mL collagenase (Roche) and 20 U/ml DNaseI (Sigma, St. Louis, MO) in RPMI 1640 + 10% FCS for 60 min at 37 °C. Tissues were further dispersed through an 18-gauge needle (5-ml syringe), RBCs were lysed and samples were filtered twice through 100-μm nylon mesh. Cells were resuspended in PBS. Live cells were identified using LIVE/DEAD Fixable Yellow Dead Cell Stain kit (Thermo Fisher Scientific, Waltham, MA), then washed and resuspended in PBS with 1% FCS. Fc receptors were blocked with purified anti-CD16/ 32 (clone 93; BioLegend, San Diego, CA). Surface markers were identified using Abs (clones) against the following antigens, all from BioLegend: anti-Cd11c (N418), MHC II (M5/114.15.2), Cd11b (M1/70), OX-40L (RM134L), Cd3 (145–2C11), Cd4 (GK1.5), Cd8 (53–6.7), Cd25 (3C7), Cd90 (53–2.1), cKit (2B8), ST2 (D1H9), Gr-1 (RB6- 8C5), B220 (RA3–6B2), Ter119 (Ter-119). For innate lymphoid cell staining, lineage markers were anti-CD3, CD11b, B220, Gr-1, and TER119. ILC2: CD45+ /Lin-/ CD90+ / ST2+. Data was collected using a NovoCyte flow cytometer (ACEA Bioscience, Inc. San Diego, California). Data analysis was performed using FlowJo software (Tree Star, Oregon, USA).
Malinczak C.A., Fonseca W., Rasky A.J., Ptaschinski C., Morris S., Ziegler S.F, & Lukacs N.W. (2019). Sex-associated TSLP-induced immune alterations following early-life RSV infection leads to enhanced allergic disease. Mucosal Immunology, 12(4), 969-979.
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9

Multi-tissue Single Cell Analysis

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Single cell suspensions were prepared from spleen, BM, or thymus, followed by red blood cell lysis (for spleen and BM only) (R7757; Sigma-Aldrich). The following antibodies were from BioLegend: anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), anti-CD11b (clone M1/70), anti-CD19 (clone 6D5), anti-CD48 (clone HM48-1), anti-CD150 (clone TC15-12F12.2), anti-Ly6C/G (Gr-1) (clone RB6-8C5), anti-B220 (clone RA3-6B2), anti-Ly6A/E (Sca-1) (clone D7), anti-Ter119 (clone TER-119), anti-CD41 (clone MWReg30), anti-CD105 (clone MJ7/18), anti-CD16/32 (clone 93), anti-CD43 (clone S11), anti-IgM (clone AF6-78), anti-CD93 (clone AA4.1), anti-CD44 (clone IM7), anti-cKit/CD117 (clone 2B8), and anti-Lineage, with an antibody cocktail containing the following: anti-CD3e (clone 17A2), CD4 (clone GK1.5), CD8 (clone 53-6.7), TCRβ (clone H57-597), TCRγδ (clone GL3), NK1.1 (clone PK136), CD11b (clone M1/70), CD11c (clone N418), Ter119 (clone TER-119), Gr-1 (clone RB6-8C5), B220 (clone RA3-6B2), and CD19 (clone 6D5). Dead cells were excluded with Zombie Aqua Fixable Viability Dye (BioLegend). Cells were analyzed using a BD LSRFortessa flow cytometer (Becton Dickinson) and data were analyzed with FlowJo (TreeStar/BD).
Graniel J.V., Bisht K., Friedman A., White J., Perkey E., Vanderbeck A., Moroz A., Carrington L.J., Brandstadter J.D., Allen F., Shami A.N., Thomas P., Crayton A., Manzor M., Mychalowych A., Chase J., Hammoud S.S., Keegan C.E., Maillard I, & Nandakumar J. (2021). Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline. Life Science Alliance, 5(1), e202101208.
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10

ILC Identification and Sorting Protocol

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Antibodies against the following molecules were used to identify ILC populations: CD45 (clone 30-F11, Biolegend), GATA-3 (TWAJ, eBioscience) and RORγt (B2D, eBioscience). Lineage marker-expressing cells were excluded using a cocktail of FITC-conjugated antibodies purchased from Biolegend against B220 (RA3-6B2), CD3ε (145-2C11), CD4 (RM4-5), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), CD19 (6D5), CD49b (DX5), FcεRIα (MAR-1), Gr-1 (RB6-8C5), NKp46 (29A1.4), TCRβ (H57-597) and TCRγδ (UC7-13D5). Intracellular staining was performed using the Foxp3/Transcription Factor staining kit (eBioscience). Cultured ILC2 were surface stained with Thy1.2 (53-2.1, BD Biosciences), Sca-1 (D7, Biolegend), c-Kit (2B8, Biolegend) and lineage markers, then sorted on a BD FACSAria II on the basis of Lin− Thy1+ Sca-1+ and c-Kitlow/−. Dead cells were excluded from all experiments based on staining with eFluor 780 fixable viability dye (eBioscience), non-specific Fcγ receptor interactions were blocked with anti-CD16/32 (clone 93, Biolegend), and analyses were restricted to single cells by FSC-H and FSC-W signals. CountBright beads (ThermoFisher) were used to determine cellularity. Cells were analyzed on an LSRII Fortessa or a FACSCalibur cytometer (BD Biosciences) and data were processed using FlowJo version 10.0.8 (Tree Star Inc.).
Burton O.T., Medina-Tamayo J., Stranks A.J., Miller S., Koleoglou K.J., Weinberg E.O, & Oettgen H.C. (2018). IgE promotes type 2 innate lymphoid cells in murine food allergy. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 48(3), 288-296.
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