HLT cells were cultured in a round-bottom 96-well plate containing 20 μM of cepharanthine, ergoloid, ciclesonide, hydroxychloroquine, ivermectin, or camostat mesylate alone or in combination with the stimuli LPS (50 ng/ml) and IFN-γ (100 ng/ml). For each patient, a negative control, cells treated with only medium, and a positive control, cells incubated in the presence of LPS and IFN-γ, were included. Immediately, brefeldin A (BD Biosciences) and monensin (BD Biosciences) were added to cells and cultured overnight at 37°C in 5% CO2. Next day, cellular suspensions were stained with the following antibodies: anti-CD11b (FITC, BioLegend), anti-CD69 (PE-CF594, BD Biosciences), anti-CD14 (APC-H7, BD Biosciences), anti-EpCAM (APC, BioLegend), anti-CD3 (BV650, BD Biosciences), anti-CD45 (BV605, BioLegend), and anti-HLA-DR (BV421, BioLegend). Cells were subsequently fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) and intracellularly stained with anti-IL-6 (PE-Cy7, BioLegend), and anti-CXCL10 (PE, BioLegend). Cell viability was determined using an AQUA viability dye for flow cytometry (LIVE/DEAD fixable AQUA, Invitrogen). After fixation with PBS 2% PFA, cells were acquired in an LSR Fortessa (BD Biosciences), and data were analyzed using the FlowJo v10.6.1 software (TreeStar).
Anti cd14 apc h7
Manufactured by BD
Sourced in United States
Anti-CD14 APC-H7 is a fluorescently-labeled antibody that binds to the CD14 antigen, a protein expressed on the surface of various immune cells. It can be used in flow cytometry applications to identify and quantify CD14-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8 percp cy5, by BD (4 mentions)
Anti cd19 apc h7, by BD (3 mentions)
Anti cd4 bv650, by BD (3 mentions)
Anti cd3, by BD (2 mentions)
Anti cd3 percp cy5, by BD (2 mentions)
Anti cd45ra fitc, by BD (2 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti epcam apc, by BioLegend (1 mentions)
Anti cd11b fitc, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Brefeldin a, by BD (1 mentions)
Monensin, by BD (1 mentions)
Cytofix cytoperm solution kit, by BD (1 mentions)
Anti cd107a fitc, by BD (1 mentions)
Facsverse flow cytometer, by FlowJo (1 mentions)
Live dead bv510, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ pe cy7, by BD (1 mentions)
Anti ifn γ, by BD (1 mentions)
10 protocols using anti cd14 apc h7
1
Cytokine Response of Immune Cells to Antiviral Compounds
2
Immunophenotyping of Granulocytic Myeloid-Derived Suppressor Cells
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All antibodies were purchased from Biolegend (San Diego, CA, USA), except for anti‐CD14‐APCH7, anti‐CD3‐PerCPCy5.5, anti‐CD107a‐FITC and anti‐IFN‐γ‐PECy7, which were purchased from BD Biosciences (Franklin Lakes, NJ, USA). To determine the frequencies of gMDSCs, peripheral blood mononuclear cells were first stained with live/dead‐BV510 (Life Technologies, Waltham, MA, USA) and surface antibodies and then incubated for 20 minutes at 4°C in the dark. gMDSCs were counted as CD15+HLA−DR−CD14− cells derived from CD33+CD11b+ myeloid cells. For intracellular staining, cells were permeabilized with the Cytofix/Cytoperm Solution Kit (BD Bioscience) and stained with anti‐arginase I‐FITC (R&D, Minneapolis, MN, USA) or anti‐IFN‐γ‐PECy7; a matched isotype antibody was used as a negative control. After staining, cells were fixed in 1% paraformaldehyde and analysed using a FACS Verse flow cytometer and FlowJo software. For morphologic analysis, flow cytometric‐isolated gMDSCs were stained with a Wright‐Giemsa kit (Solarbio, Beijing, China) following the manufacturer's instructions.
3
Comprehensive Immunophenotyping of Myeloid-Derived Suppressor Cells
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Evaluation of MDSC percentage was accomplished with 0.5 × 106 PBMCs stained with anti-CD15 FITC, anti-CD33 PE, anti-HLA-DR PERCP, cocktail of antibodies anti-CD3, -CD56, -CD19 (Lin) APC, anti-CD14 APC-H7, anti-CD11b PE-C7, anti-CD16 Pacific Blue (BD Biosciences). The frequency of hematopoietic progenitors was evaluated on 1 × 10∧5 viable cells by using Lineage cocktail 1 FITC, anti-CD34 APC, anti-CD45 V500, 7AAD (BD Biosciences), and anti-CD38 PE-Vio770 (Miltenyi Biotec). Intracellular flow cytometry was performed by using anti-CD3, anti-CD8, and anti-IFN-γ (BD Biosciences, USA). Acquisition of 100,000 events was performed in the leukocyte-gated population on FACS CANTO II and analyzed with FACS DIVA software (BD Biosciences).
4
Tetramer-Based Enrichment of Antigen-Specific T Cells
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PBMCs (2.4–10 × 106) were stained with WT HLA-A2S269–277-PE and variant HLA-A2S269–277-APC tetramers at room temperature for 1 h in MACS buffer (PBS with 0.5% BSA and 2 mM EDTA). Cells were then incubated with anti-PE and anti-APC microbeads (Miltenyi Biotec), and tetramer+ cells were enriched using magnetic separation (9 (link)). Lymphocytes were stained with anti-CD71-BV421 (#562995), anti-CD4-BV650 (#563875), anti-CD27-BV711 (#563167), anti-CD38-BV786 (#563964), anti-CCR7-AF700 (#561143), anti-CD14-APC-H7 (#560180), anti-CD19-APC-H7 (#560177), anti-CD45RA-FITC (#555488), anti-CD8-PerCP-Cy5.5 (#565310), anti-CD95-PE-CF594 (#562395), anti-PD1-PE-Cy7 (#561272) (BD Biosciences), anti-CD3-BV510 (#317332), anti-HLA-DR-BV605 (#307640) (BioLegend), and LIVE/DEAD near-infrared stain (#L10119, Invitrogen) for 30 min, fixed with 1% PFA before acquiring data on an LSRII Fortessa (BD Bioscience). FCS files were analyzed using FlowJo v10 software.
5
Multiparametric Phenotyping of Antigen-specific T Cells
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Approximately 20–50 million cryopreserved peripheral blood mononuclear cells were thawed and incubated with anti-human FcR block (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min on ice. Cells were then stained with PE- and/or APC-tetramer for 1 h, washed once, then incubated with anti-PE and/or anti-APC microbeads (Miltenyi Biotec) before passing through a LS column (Miltenyi Biotec) to enrich for tetramer+ cells as previously described.30 (link), 31 (link) Cells were then surface stained with human anti-CD3 BV570 (#300436, Biolegend, San Diego, CA, USA), anti-CD8 PerCP-Cy5.5 (#565310, BD Biosciences, San Jose, CA, USA), anti-CD14 APC-H7 (#560180, BD Biosciences), CD19 APC-H7 (#560177, BD Biosciences), anti-CD45RA FITC (#555488, BD Biosciences), anti-CD27 AF700 (#56027942, eBioscience, San Diego, CA, USA) and LIVE/DEAD Fixable Aqua (Molecular Probes, Eugene, OR, USA) before fixing with 1% paraformaldehyde. Cells were acquired using the LSR Fortessa II (BD Biosciences) and analyzed by FlowJo software (Treestar, Inc., Ashland, OR, USA).
6
Generating CD8+ T Cell Lines from PBMCs
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CD8+ T cell lines were generated as previously described25 (link),58 (link). In brief, PBMCs were incubated with 1 μM of individual peptide (NQK-Q8 or NQK-A8) and cultured for 10–14 days in RPMI-1640 supplemented with 2 mM MEM non-essential amino acid solution (Sigma-Aldrich), 100 mM HEPES (Sigma-Aldrich), 2 mM l -glutamine (Sigma-Aldrich), penicillin–streptomycin (Life Technologies), 50 mM 2-ME (Sigma-Aldrich) and 10% heat-inactivated fetal bovine serum (Bovogen). The cultures were supplemented with 10 IU IL-2 2–3 times weekly. CD8+ T cell lines were used fresh for subsequent analysis. For the double tetramer staining experiments 0.5 × 106 cells from the CD8+ T cell lines were stained with a single PE-conjugated tetramer (HLA-B*15:01-NQK-Q8 or HLA-B*15:01-NQK-A8) or double stained with both tetramers (PE-conjugated NQK-A8 and APC-conjugated NQK-Q8 tetramer) for 1 h at room temperature. Cells were washed and surface-stained with anti-CD3-BV480 (dilution 1:100), anti-CD8-PerCP-Cy5.5 (1:50), anti-CD4-BV650 (1:100), anti-CD14-APCH7 (1:200) and anti-CD19-APCH7 (1:100) antibodies (all BD Biosciences) and Live/Dead Fixable Near-IR Dead Cell Stain (Life Technologies). Cells were single-cell sorted into PCR plates (Eppendorf) using the BD Aria Fusion system.
7
Comprehensive Immune Cell Profiling by Flow Cytometry
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NK, T and Monocyte cell surface antigens were analysed by flow cytometry. Briefly, two million PBMCs were stained with a surface antibody’s cocktail: anti-CD3 PerCP-Cy 5.5, anti-CD56 APC and anti-CD16 PE-CyTM7 from BD Biosciences, anti-CD45 Krome Orange from Beckman Coulter and anti-Vδ2 Brilliant Violet 605 from Biolegend (Mix NK and gammadelta T cells); anti-CD4 FITC, anti-CD8 PE and anti-CD3 Pacific Blue from BD Biosciences and anti-CD45 Krome Orange (Mix T cells); anti-CD14 APC-H7 and anti-CD16 V450 from BD Biosciences and anti-CD45 Krome Orange (Mix Monocytes), in 1× PBS, 1% Bovine Serum Albumin (BSA) (from Sigma Aldrich) and 0.1% Sodium azide (NaN3) (from Serva) solution for 15 min at 4°C. Afterwards, PBMCs were washed with 0.3 mL of 1× PBS, 1% BSA and 0.1% NaN3 solution and centrifuged at 1600 rpm for 5 min. Next, PBMCs were fixed with 1% Paraformaldehyde (PFA) (Bio-Rad, Hercules, California, USA) in 1× PBS, 1% BSA and 0.1% NaN3 solution for 15 min at room temperature in the dark, washed as before and centrifuged at 1600 rpm for 5 min. Cells were acquired using a FACSLyric (BD Biosciences) and analysed by BD FACSuite software (BD Biosciences).
8
CD8+ T cell Activation Assay
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Expanded A68/NP145+ CD8+ T cells were stimulated with 1 µM peptide (DATYQRTRALVR, DTTYQRTRALVR, DVTYQRTRALVR or pool of DATYQRTRALVR, DTTYQRTRALVR, and DVTYQRTRALVR) and cultured for 5 h in the presence of 10 U/ml rIL2 and Golgi Stop (BD Biosciences). Following activation, cells were surface stained for 30 min with human anti-CD3-BV510 (1:200, Biolegend #317332), anti-CD4-BV650 (1:200, BD Horizon #563875), anti-CD14-APC-H7 (1:100, BD Pharmingen #560180), anti-CD19-APC-H7 (1:100, BD Pharmingen #560177), anti-CD8-PerCPCy5.5 (1:100, BD Pharmingen #565310), Live/Dead near-infrared (1:800, Invitrogen), and PE-streptavidin-conjugated A68/NP145 (DATYQRTRALVR) tetramer. Cells were fixed with BD Fix-Perm buffer (BD Biosciences) for 20 min, before intracellular staining for 30 min with human anti-IFN-γ-V450 (1:100, BD Horizon #560371) in perm wash buffer (BD Biosciences). Cells were washed, acquired on the BD Fortessa (BD Biosciences) and analyzed using the Flowjo software (Treestar, OR, USA).
9
Monoclonal Antibody Characterization of MoDC and γδ T Cells
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The following monoclonal antibodies were used to characterize MoDC: anti-CD86 FITC, anti-CD1a PE, anti-HLA-DR PERCP, anti-CD83 APC, anti-CD14 APC-H7, anti-CD80 FITC, anti-CD40 PE, anti-HLA-I APC, anti-CCR7 PE-Cy7, anti-CCR5 APC-H7 from BD Biosciences, and anti-BT3A.1 PE from BioLegend. To evaluate γδ T lymphocytes phenotype we used anti-Vδ2 FITC, anti-CD3 PE, anti-CD69 PERCP, anti-CD45RA PE-Cy7, anti-CD27 APC, anti-CD16 PACIFIC BLU, anti-CD25 APC-H7 (BD Biosciences). In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide, and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 4% paraformaldehyde, and analyzed using a FACS Canto II (Becton Dickinson). Since the presence of 2 purified populations, the gating strategy was performed as follow: dead cells were excluded by scatter characteristics; MoDC were identified by morphological parameters (FSC vs SSC); gated cells were then analyzed for the expression of the molecules described above. T lymphocytes were first gated by using morphological parameters; in this gate Vγ9Vδ2 T cells were identified as Vδ2+CD3+. Analysis was carried out by using Facs Diva software (Becton Dickinson). The histogram overlays were performed by FlowJo software (TreStar, Olten, Switxìzerland).
10
Flow Cytometry Analysis of ASC
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PBMCs were analyzed with a simple standard flow cytometry panel at start and after 7 days of culturing to identify antibody-secreting cells (Supplementary Figure 2 Supplementary Figure 2
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