Flow cytometry was performed using an LSRFortessa (BD) and analyzed on FlowJo v10 (BD). Fc receptors were blocked using anti-CD16/32 (clone 2.4G2, Bio X Cell). Antibodies specific for mouse and human antigens (Supp. Table S1 ) conjugated to various fluorochromes were used to stain single cells. If necessary, cells were fixed using the eBioscience Fixation and Permeabilization Buffer Kit (cat# 88–8824-00) as per the manufacturer’s protocol. Intracellular cytokine staining was performed with the BD Cytofix/Cytoperm Kit (cat# 554714), as per the manufacturer’s protocol. For intracellular IFNγ and TNF staining, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich, cat# 79346) and 1 μM ionomycin (Life Technologies, cat# I24222) and 1 μg/mL LPS (Sigma-Aldrich, cat# L6529) and incubated at 37oC, 5% CO2 in the presence of 10 μg/mL brefeldin A (Affymetrix, Cat# 00–4506-51) for 4 hours.
Anti cd16 32 clone 2.4g2
Manufactured by BioXCell
Anti-CD16/32 (clone 2.4G2) is a monoclonal antibody that binds to the mouse CD16 and CD32 receptors. It is commonly used in flow cytometry and other immunological applications to block Fc receptor-mediated binding.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Fixation and permeabilization buffer kit, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Intracellular fixation buffer, by Thermo Fisher Scientific (1 mentions)
Brilliant violet 421, by BioLegend (1 mentions)
Facscanto 2 hts, by BD (1 mentions)
Brdu flow kit, by BD (1 mentions)
Hanks buffered saline, by Merck Group (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Collagenase d, by Roche (1 mentions)
Dnase 1, by Roche (1 mentions)
Bromodeoxyuridine (brdu), by BD (1 mentions)
Zombie aqua fixable dye, by BioLegend (1 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
Lung dissociation kit, by Miltenyi Biotec (1 mentions)
5 protocols using anti cd16 32 clone 2.4g2
1
Multiparametric Flow Cytometry Analysis
2
Phospho-PD-1 Expression in Mouse Cells
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One microgram of anti-CD16/32 (clone 2.4G2, BioXCell) per 100 μL was first added to mouse cells to block Fc receptors. Staining was performed for 30 minutes on ice, protected from light. After staining, cells were fixed and permeabilized using an Intracellular Fixation Buffer (eBioscience #88–8824–00) for 30 minutes on ice and then stained with phospho–PD-1 mAb Brilliant Violet 421 (10 μg/mL; clone 407.6G12, custom conjugated by Biolegend) on ice for 30 minutes. Isotype control and Fluorescence Minus One control were used for gating. Samples were analyzed on an LSR II or a FACSCanto II HTS (BD Biosciences) and analyzed using FlowJo software (TreeStar). A sample gating strategy can be found in Supplementary Fig. S1A.
3
Mammary Gland Macrophage Isolation
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Unless otherwise stated, the 4th MG from virgin females was collected, inguinal lymph node was removed and the fat pad was finely minced with scissors in Hank’s Buffered Saline (Sigma-Aldrich, H9394). MG was digested with 1 mg/ml collagenase D (Roche, 1108886601) and 50 µg/ml DNase 1 (Roche, 10104159001) for 60 min at +37 °C. Cell suspension was filtered through silk (pore size 77 µm). The cell suspensions were incubated with purified anti-CD16/32 (clone 2.4G2; Bio X Cell) for 10 min on ice to block Fc-receptors. Immunofluorescence stainings were performed at 4 °C for 20 min (the antibodies used are listed Supplementary Table 1 ). Flow cytometry was performed with a LSR Fortessa flow cytometer (Becton Dickinson), and data were analyzed using the FlowJo software (FlowJo LLC).
For proliferation analysis, mice were injected i.p. with 120 μl of 10 mg/ml solution of bromodeoxyuridine (BrdU, BD Bioscience) 2 h before the sacrification. After immunofluorescence stainings cells were fixed, and stained with FITC–conjugated BrdU antibody (BrdU Flow Kit, BD Bioscience).
For qPCR analysis, CD45+Siglec-F−CD11b+F4/80Int and CD45+Siglec-F−CD11b+F4/80Hi or CD45+Siglec-F−CD11b+F4/80+CD206Neg/low and CD45+Siglec-F−CD11b+F4/80+CD206Hi cells from MG of 5 wk old mice were sorted using a FACS aria II cell sorter (100 µm nozzle, Beckton-Dickinson). The purity of the isolated populations was >95%.
For proliferation analysis, mice were injected i.p. with 120 μl of 10 mg/ml solution of bromodeoxyuridine (BrdU, BD Bioscience) 2 h before the sacrification. After immunofluorescence stainings cells were fixed, and stained with FITC–conjugated BrdU antibody (BrdU Flow Kit, BD Bioscience).
For qPCR analysis, CD45+Siglec-F−CD11b+F4/80Int and CD45+Siglec-F−CD11b+F4/80Hi or CD45+Siglec-F−CD11b+F4/80+CD206Neg/low and CD45+Siglec-F−CD11b+F4/80+CD206Hi cells from MG of 5 wk old mice were sorted using a FACS aria II cell sorter (100 µm nozzle, Beckton-Dickinson). The purity of the isolated populations was >95%.
4
Phenotyping Macrophage Subsets
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BMDMs were stained for viability using the Zombie Aqua Fixable dye (BioLegend), followed by Fc blocking using 20 µg/ml anti-CD16/32 (clone 2.4G2; BioXCell) for 20 min. Cells were stained with BV421-conjugated anti-CD64 (clone X54-5/7.1; BioLegend) and acquired on a BD LSRII. Data were analyzed using FlowJo v10 (BD Biosciences).
5
Single-cell RNA-Seq of IL-4-expressing CD4+ T Cells
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At day 10 after Nb infection lungs and MLNs of IL-4eGFP reporter (4get) mice were harvested.
Lungs were perfused with PBS, cut into small pieces and digested with the commercial "Lung Dissociation kit" (Miltenyi, Bergisch Gladbach, GER) according to manufacturer's instructions.
Digested lungs and complete MLNs were gently mashed through a 100 μm cell strainer. For lung cells a 40% percoll purification was applied and erythrocytes were lysed with ACK-buffer (0.15 M NH4Cl, 1 mM KHO3, 0.1 mM Na2EDTA). Then samples of both organs were treated with Fcreceptor blocking antibody (anti-CD16/32, clone 2.4G2, BioXCell, West Lebanon, NH) and stained with anti-CD4-Percp-Cy5.5 antibody (clone: RM4-5). IL-4eGFP + CD4 + cells were sorted and for each sample, 5000 cells were subjected to 10x Chromium Single Cell 5′ Solution v2 library preparation using the TCR-specific VDJ library kit according to manufacturer's instructions (10xGenomics, Pleasanton, CA). Gene expression libraries were sequenced on an Illumina HiSeq 2500 sequencer using the recommended read lengths for 10x Chromium 5' v2 chemistry to a depth of at least 30000 reads per cell. VDJ libraries were sequenced as paired 150 bp reads to a depth of at least 30000 reads per cell.
Lungs were perfused with PBS, cut into small pieces and digested with the commercial "Lung Dissociation kit" (Miltenyi, Bergisch Gladbach, GER) according to manufacturer's instructions.
Digested lungs and complete MLNs were gently mashed through a 100 μm cell strainer. For lung cells a 40% percoll purification was applied and erythrocytes were lysed with ACK-buffer (0.15 M NH4Cl, 1 mM KHO3, 0.1 mM Na2EDTA). Then samples of both organs were treated with Fcreceptor blocking antibody (anti-CD16/32, clone 2.4G2, BioXCell, West Lebanon, NH) and stained with anti-CD4-Percp-Cy5.5 antibody (clone: RM4-5). IL-4eGFP + CD4 + cells were sorted and for each sample, 5000 cells were subjected to 10x Chromium Single Cell 5′ Solution v2 library preparation using the TCR-specific VDJ library kit according to manufacturer's instructions (10xGenomics, Pleasanton, CA). Gene expression libraries were sequenced on an Illumina HiSeq 2500 sequencer using the recommended read lengths for 10x Chromium 5' v2 chemistry to a depth of at least 30000 reads per cell. VDJ libraries were sequenced as paired 150 bp reads to a depth of at least 30000 reads per cell.
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