At designated time points, DLNs, lungs, spleens, or tumor tissues were collected, ground, and filtered through nylon mesh filters (40 μm) and washed with PBS containing 1% FBS, respectively. The single-cell suspensions were blocked with anti-CD16/32 (BioLegend) for 30 min on ice and then stained with fluorescence-labeled anti-mouse CD45 (30-F11), CD3e (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), Ly-6G/Ly-6C (RB6-8C5), F4/80 (BM8), CD62L (MEL-14), and CD44 (IM7) (all from BioLegend; dilution, 1:100) for 30 min on ice. For DC characterization, DC suspensions were blocked with anti-CD16/32 (BioLegend) for 30 min on ice and then stained with fluorescence-labeled anti-mouse CD11c (N418), CD80 (16-10A1), and CD86 (GL-1) for 30 min on ice. For intracellular protein staining, the samples were blocked with anti-CD16/32 (BioLegend) for 30 min on ice and then stained with anti-mouse CD4 or CD8 for 30 min on ice, respectively. Subsequently, cells were fixed, permeabilized, and stained with anti-mouse Foxp3 (FJK-16s) or IFN-γ (XMG1.2) at room temperature for 30 min, respectively. Last, all samples were washed with PBS containing 1% FBS two times. All the flow cytometric analysis was conducted using the LSRFortessa Cell Analyzer (BD Biosciences), and data analysis was carried out using FlowJo software (www.flowjo.com ; Tree Star).
Anti cd16 32
Manufactured by BioLegend
Sourced in United States
Anti-CD16/32 is a laboratory reagent used for the detection and analysis of CD16 and CD32 proteins in various biological samples. It is a monoclonal antibody that specifically binds to these cell surface receptors, which are involved in immune cell function and signaling. The core function of this product is to facilitate the identification and quantification of cells expressing CD16 and CD32 through techniques such as flow cytometry.
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Lab products found in correlation
Anti cd45, by BioLegend (8 mentions)
Anti cd4, by BioLegend (7 mentions)
Anti ter119, by BioLegend (6 mentions)
Anti cd19, by BioLegend (5 mentions)
Anti cd11b, by BioLegend (5 mentions)
Dnase 1, by Merck Group (4 mentions)
Anti cd45 fitc, by BioLegend (4 mentions)
Ionomycin, by Merck Group (4 mentions)
Anti cd3, by BioLegend (4 mentions)
Anti cd11b v450, by BD (4 mentions)
Anti cd45 apc cy7, by BD (4 mentions)
Perm wash buffer, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Anti cd4 apc, by BioLegend (4 mentions)
Facsaria 2, by BD (4 mentions)
Lsrfortessa, by BD (4 mentions)
Anti b220, by BioLegend (3 mentions)
Anti sca 1, by BioLegend (3 mentions)
Anti cd150, by BioLegend (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
115 protocols using anti cd16 32
1
Comprehensive Immune Cell Profiling
2
Isolation and Purification of Mouse Lung Endothelial Cells
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Mouse lung EC were isolated from three to four pairs of lungs dissected from control or Sptlc1 ECKO adult mice 4 weeks post tamoxifen treatment. Freshly isolated lung tissues were minced with scissors and allowed to digest at 37 °C in Liberase (0.6 U/mL, Sigma) in HBSS for 45 min. Samples were further subjected to mechanical disruption by gentleMACS Octo Dissociator (Miltenyi Biotec) and filtration through fine steel mesh (40 µm, Falcon). Cells were washed twice with HBSS containing 0.5% Fatty Acid Free BSA. RBC were removed by ACK lysis buffer (Thermo Fisher). Cells were incubated with CD45 micro beads (Miltenyi Biotec) at 4 °C for 15 min to deplete CD45+ cells. Remaining cells were incubated with CD31 micro beads (Miltenyi Biotec) at 4 °C for 15 min to enrich EC. The purity of each population was determined by flow cytometry.
Cells were stained with stained with phycoerythrin (PE)-conjugated anti-mouse CD31 (BioLegend, 1:200), allophycocyanin (APC)-conjugated anti-mouse CD45 (BioLegend, 1:200) in blocking solution (0.25% FAF-BSA in HBSS) with anti-CD16/32 (BioLegend, 1:200) for 15 min on ice. Stained cells were washed with HBSS containing 10% serum twice and analyzed by BD FACSCalibur Flow Cytometer (BD Biosciences). CD31 and CD45 were gated as shown inFigure 1B .
Cells were stained with stained with phycoerythrin (PE)-conjugated anti-mouse CD31 (BioLegend, 1:200), allophycocyanin (APC)-conjugated anti-mouse CD45 (BioLegend, 1:200) in blocking solution (0.25% FAF-BSA in HBSS) with anti-CD16/32 (BioLegend, 1:200) for 15 min on ice. Stained cells were washed with HBSS containing 10% serum twice and analyzed by BD FACSCalibur Flow Cytometer (BD Biosciences). CD31 and CD45 were gated as shown in
3
Isolation and Characterization of Synovial Fibroblasts
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Isolation of SFs was performed from both hind paws. The ankle joints were dissected, and the tissues were disaggregated by incubation for 30 min at 37 °C in an enzymatic digestion medium consisting of DMEM, 10%heat-inactivated FBS, collagenase (0.5 mg ml−1) from Clostridium histolyticum (Sigma, C5138) and 0.03 mg ml−1 DNase (Sigma, 9003-98-9). Upon washing the cells with PBS containing DNase, they were blocked in 1% BSA in PBS and Fc blocker (unlabelled anti-CD16/32, Biolegend 101302) for 10 min at 4 °C and stained with fluorophore-conjugated antibodies for 20 min at 4 °C (anti-Pdpn PE-Cy7, Biolegend 127411; anti-Thy1 A647, Biolegend 105318; anti-CD31 APC/Fire 750, Biolegend 102433; anti-CD45 APC-Cy7, Biolegend 103116; anti-Ter119 APC-A780, eBioscience 47-5921-80). After washing with PBS, cells were resuspended in FACS buffer (PBS, 1%BSA). Sorting of cells was performed with BD FACSAria III and the BD FACSDiva software, and dead cells were excluded by DAPI staining. Sorting gaiting for single-cell RNA-seq/ATAC-seq and bulk RNA-seq was different (Additional file 1 : Fig. S1B). For sorted populations, purity and viability were determined by reanalysis for the target population based on cell surface markers immediately post-sorting. Purity was > 99% for each target population.
4
Murine Bone Marrow Cell Isolation and Immunophenotyping
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Bone marrow was isolated from tibia and femur of mice and erythrocytes were lysed. To sort pre-progenitor cells staining for linage markers anti-CD3e (Cat # 45-0031-80, eBioscience, 1:50), anti-GR1 (Cat # 108428, BioLegend, 1:400), anti-Mac-1 (Cat # 101228, BioLegend, 1:400), anti-CD4 (Cat # 101228, BioLegend, 1:200), anti-CD8a (Cat # 100734, BioLegend, 1:100), anti-TCRb (Cat # 109228, BioLegend, 1:100), anti-NK1.1 (Cat # 45-5941-82, eBioscience, 1:100), anti-B220 (Cat # 103236, BioLegend, 1:100) and anti-CD19 (Cat # 115534, BioLegend, 1:200) was performed. Additionally anti-CD16/32 (Cat # 101305, BioLegend, 1:200), anti-CD41 (Cat # 133906, BioLegend, 1:100), anti-Sca-1 (Cat # 108114, BioLegend, 1:200), anti-c-kit (Cat # 47-1171-82, Invitrogen, 1:50), anti-CD105 (Cat # 120412, BioLegend, 1:50) and anti-CD150 (Cat # 115910, BioLegend, 1:400) were stained to further classify cells. To sort immature granulocytes, mature granulocytes and monocytes anti-Ly6G/Ly6C (Cat # 17-5931-82, Invitrogen, 1:100), anti-CD11b (Cat # 25-0112-82, eBioscience, 1:100) and anti-CD115 (Cat # 11-1031-85, eBioscience, 1:100) were used. Gating strategy was performed as shown in Supplementary Fig 5 .
5
Murine Dendritic Cell Culture Protocol
6
Tumor Dissociation and Flow Cytometry
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Three weeks after implantation, tumors were dissected from the subcutaneous of mice, and treated with collagenase type IV (200 U/ml; Gibco) and DNase I (50 μg/ml; Sigma) for 20 min at 37 °C. Cells were passed through a 70-μm filter to remove clumps, diluted in medium, and a small aliquot taken directly for flow cytometry. Single-cell suspensions were blocked with anti-CD16/32 (1:100 dilution; Biolegend) for 20 min on ice and then incubated with appropriate antibodies (1:100 dilution) for 30 min on ice. Flow cytometry was performed on an LSRII (BD Biosciences) and data were analyzed using FlowJo software v 10.4.2 (FlowJo).
7
Molecular Mechanisms of CRAC Channel Regulation
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All plasmids used in this study were sequenced to confirm their identity. Crbn, Ampk, Slo, or HA-Ubiquitin constructs used in this study were described previously26 (link). Orai1 (MMM1013-202764440), Orai2 (MMM1013-202859855), and Orai3 (MMM1013-202842392) cDNA were purchased from Open Biosystems to construct expression vectors by a PCR-based strategy. Glutamine synthetase (GS) cDNA were synthesized from mRNA of the testis of mice and GS expression vectors were generated. The complete list of all primer sequences used in the study is provided as a Supplementary table. Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.
8
Immune Profiling of Tumor-Draining Lymph Nodes
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Tumors and cervical lymph nodes harvested from mice were collected and homogenized into the single-cell suspensions. Then, the cells were blocked with anti-CD16/32 (BioLegend, catalog no. 101302) antibodies to avoid nonspecific adsorption and stained with the following antibodies according to the manufacturer’s instructions: fluorescein isothiocyanate (FITC)–anti-CD45 (BioLegend, catalog no. 147710), Peridinin-Chlorophyll-Protein Complex (PerCP)-CD11c (BioLegend, catalog no. 117326), allophycocyanin (APC)–anti-CD80 (BioLegend, catalog no. 104714), phycoerythrin (PE)–anti-CD86 (BioLegend, catalog no. 105106), PerCP–anti-CD3 (BioLegend, catalog no. 100326), APC–anti-CD4 (BioLegend, catalog no. 100412), PE–anti-CD8 (BioLegend, catalog no. 100708), and FITC–anti-Ki67 (BioLegend, catalog no. 151212). A total of 1.0 × 105 events in the ungated flow chart in each sample were collected using a C6 plus flow cytometer and analyzed by FlowJo software (Ver. 10.0.7). The gating strategy is given in fig. S23.
9
Tumor Cell Isolation and Gene Expression
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Cell suspension (described above) was stained with Zombie Fixable Viability Kit followed by incubation with anti-CD16/32 and staining with anti-CD45 (clone 30-F11) and anti-CD31 (clone 390); all from Biolegend. At least 50,000 tumor cells (CD45neg,CD31neg,GFP+) were sorted per sample. RNA was extracted using RNeasy Plus Mini Kit (Qiagen) and cDNA was synthesized using Omniscript RT Kit (Qiagen). qPCR was performed with KAPA SYBR FAST quantitative PCR (qPCR) Master Mix (KAPA Biosystems) and analyzed in a CFX96 Touch Real-Time PCR Detection System (Biorad).
10
Comprehensive Immune Cell Profiling
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Single cell suspensions were stained with fixable viability dye eFluor® 780 (eBioscience) for 30 min at 4°C, and Fc receptors were blocked with anti-CD16/32 (BioLegend) blocking antibody prior to surface staining with antibodies. Antibodies for surface staining used are listed, mouse: CD45 (30-F11, BioLegend), TCR β (H57-597, BioLegend), CD69 (H1.2F3, eBioscience), CD1d tetramer (NIH); human: CD45 (HI30, eBioscience), TCRα/β (IP26, BioLegend), CD69 (FN50, BioLegend), Vα24 (6B11, BD Bioscience). For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend). For Ki67 staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (eBioscience), and further stained with Ki67 (SolA15, eBioscience). Data were acquired using LSR II (BD Biosciences) and analyzed using the FlowJo software (BD Biosciences).
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