The following antibodies were used for flow cytometric analysis: Anti-Perforin-Alexa fluor 488, anti-CD28-allophycocyanin (APC), anti-CD19-APC, anti-CD3-APC, anti-CD56-APC, anti-CD3-APC-cyanin7 (Cy7), anti-CD8-APC-Cy7, anti-CD14-APC-Cy7, anti-CD14-fluorescein isothiocyanate (FITC), anti-HLA-DR-FITC, anti-CD16-R-phycoerythrin (PE), anti-GATA3-PE, anti-CD45RA-PE-cyanin 5 (Cy5), anti-CD4-PE-Cy5, anti-CD16-PE-Cy5, anti-CCR7-PE-Cy7, anti-CD4-PE-Cy7, anti-CD4-V450, anti-GranzymeB-V450, anti-CD8-V500, anti-CD3-V500 (all from BD Bioscience, Franklin Lakes, NJ), anti-CD57-FITC, anti-CD4-FITC, anti-CD85j-PE, anti-CX3CR1-PE, anti-T-bet-PE-Cy7, anti-Eomes-Peridinin chlorophyll (PerCP)-efluor710 (six from eBioscience, San Diego, CA), anti-HLA-DR-PE-Cy5, anti-IL-7Rα-V450, anti-CD57-V450 (three from BioLegend, San Diego, CA), anti-CX3CR1-FITC (MBL International Corporation, Woburn, MA). For intracellular staining of T cell lineage-specific transcription factors (T-bet, Gata3, and Eomes), granzyme B and perforin, PBMC were fixed and permeabilized with Fix/Perm buffer set (BioLegend). Stained cells were acquired by a BD LSRFortessa (BD bioscience) and analyzed by using FlowJo software (ver. 9.0 or 10.0; Tree Star, OR).
Anti cd14 apc cy7
Manufactured by BD
Sourced in Germany
The Anti-CD14-APC-Cy7 is a fluorescently-labeled antibody that targets the CD14 cell surface receptor. It is designed for use in flow cytometry applications to identify and quantify CD14-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4 pe cy7, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Anti cd127 fitc, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Facs lysing solution, by BD (2 mentions)
Anti cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd8 apc cy7, by BD (1 mentions)
Anti cd19 apc, by BD (1 mentions)
Anti granzyme b v450, by BD (1 mentions)
Anti gata3 pe, by BD (1 mentions)
Anti cd8 v500, by BD (1 mentions)
Anti ccr7 pe cy7, by BD (1 mentions)
Anti cd3 v500, by BD (1 mentions)
Anti hla dr fitc, by BD (1 mentions)
Fix perm buffer set, by BioLegend (1 mentions)
Anti cd16 pe cy5, by BD (1 mentions)
Anti cx3cr1 pe, by Thermo Fisher Scientific (1 mentions)
Anti hla dr pe cy5, by BioLegend (1 mentions)
Pe cy5 anti cd4, by BD (1 mentions)
Anti t bet pe cy7, by Thermo Fisher Scientific (1 mentions)
10 protocols using anti cd14 apc cy7
1
Multiparametric Flow Cytometry Profiling of Immune Cells
2
Differentiation and Characterization of Monocyte-Derived Immune Cells
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Monocytes were seeded in a 96-well plate at 3x105 cells per well and differentiated to MΦ1, MΦ2 and DCs as described above. After 7 days, the cells were challenged with 500ng/ml LPS (Sigma) and surface markers were determined. Therefore, the cells were detached from the plate using 5mM EDTA (20 minutes at 37°C). The cells were spun down at 300xg, the EDTA was discarded and the cells were incubated with a mix of anti-CD14-APC-Cy7, anti-CD209-PerCP-Cy5.5, anti-CD163-PE and anti-CD80-PE-Cy7 (5μl of each antibody, all antibodies from BD). The antibodies were incubated for 20 minutes at 4°C. After incubation, 200μl PBS was added and the cells were spun down at 300xg. The supernatant was discarded, 200μl PBS was added once more and the cells were collected by centrifugation. Readout was done on a BD FACS CANTO II and subsequent analysis were done using BD DIVA software. Plots were made in Sigmaplot (Systat Software).
3
Cytokine Production in Activated T Cells
4
Flow Cytometry Staining of MR1 Tetramers
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SKW-3.β2mnull.CD8αα or SKW-3.β2m null.CD8αβ (105 per sample) were stained with MR1 or MHC-I tetramers in PBS + 2% FBS for 20 min at 4°C in the dark. Cells were washed with PBS + 2% FBS and resuspended in a surface antibody stain consisting of anti-CD3-BV421 (#562426, UCHT1; BD Horizon), anti-CD8α-BUV805 (#564912, SK1; BD Horizon), anti-CD8β-APC (#641058, 2ST8.5H7; BD FastImmune), and LIVE/DEAD fixable Near-IR dead cell stain (#L10119; Thermo Fisher Scientific) for a further 20 min at 4°C in the dark. Cells were washed twice with PBS + 2% FBS and data were acquired using a BD LSR Fortessa (BD Biosciences). PBMCs were stained with MR1 tetramers as described in (Souter et al., 2019 (link)). In brief, PBMCs (107 per sample) were stained with MR1 tetramer in PBS + 2% FBS for 30 min at room temperature in the dark, washed with PBS + 2% FBS, and stained with surface antibodies anti-CD3-BV421, anti-CD19-APC-Cy7 (#302218, HIB19; Biolegend), anti-CD14-APC-Cy7 (#557831, MφP9; BD Pharmingen), anti-CD8α-BUV805, anti-CD8β-APC, anti-CD161-PE-Vio770 (#130-113-597, REA631; Miltenyi Biotec), anti-CD4-AF700 (#557922, RPA-T4; BD Pharmingen), and LIVE/DEAD fixable Near-IR dead cell stain for 20 min at 4°C. Cells were washed twice and resuspended in PBS + 2% paraformaldehyde (PFA) before data acquisition on a BD LSR Fortessa.
5
Multiparameter Flow Cytometry Analysis
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Flow cytometry analyses were performed on an LSR Fortessa flow cytometer (BD Biosciences). Automatic compensation was performed using CompBeads (BD Biosciences). Fluorescence minus one controls were performed to define gates of positivity. Following antibodies and reagents were used: biotinylated anti-CD11b, anti-CD33 PE-Cy7, anti-HLA-DR PE-Cy7, anti-CD15 FITC, anti-CD8 PE, anti-CD4 APC-Hy, anti-CD127 FITC, anti-CD16 FITC, anti-CD45 RO PE, andi-CD45 RA FITC, anti-CD95 PD-CF594, anti-PD-1 APC, anti CD62L APC (BD Biosciences); anti-CD14 APC-Cy7, anti-CD3 BV 605, anti-CD3 PE/Dazzle 594, anti-CD4 PE-Cy7, anti-CD4 PerCPCy5.5, anti-CD56 PE-Cy7, andi-CD28 FITC, anti-CD27 APC, anti-ICOS APC-Cy7, anti-CD137 PE, anti-CD137 APC, Zombie Yellow Fixable Viability Kit (BioLegend); eFluor 450 labeled streptavidin, anti-CD8 APC-H7, anti-Foxp3 APC, anti-CCR7 APC, anti-TIM3 eFluor 450, anti-TIGIT PE, anti-LAG3 PerCPeFluor710 (eBioscience), anti-CD25 PE (Myltenyi Biotec) and anti-OX40 APC (R&D Systems).
6
Ex Vivo Human Skin DC Isolation
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Ex vivo DCs were obtained from healthy human skin as described elsewhere (40 (link)). Intradermal injections were carried out with 50 μl of phosphate-buffered saline, LPS+MF, VD3 (25 μM), and liposomes DSPG or DPTAP with or without VD3 in concentrations corresponding to the soluble vitamin control. Migrating cells were stained for the skin DC markers with anti-CD11c-PE-Cy7 (eBioscience, Thermo Fisher), anti-HLA-DR-PercP (BD), anti-CD1a-FITC (BD), and anti-CD14-APC-Cy7(BD).
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Ex vivo DCs were obtained from healthy human skin as described elsewhere (40 (link)). Intradermal injections were carried out with 50 μl of phosphate-buffered saline, LPS+MF, VD3 (25 μM), and liposomes DSPG or DPTAP with or without VD3 in concentrations corresponding to the soluble vitamin control. Migrating cells were stained for the skin DC markers with anti-CD11c-PE-Cy7 (eBioscience, Thermo Fisher), anti-HLA-DR-PercP (BD), anti-CD1a-FITC (BD), and anti-CD14-APC-Cy7(BD).
7
CYNK Cell Cytotoxicity Assay for Influenza
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A549 cells were seeded at 104 cells per well in a 96-well plate in complete F-12K medium. PR8 infection was performed as described above. CYNK cells were added 24 hours post infection at an effector-to-target (E:T) ratio of 10:1 with anti-CD107a – BV786 (clone H4A3, BD) in assay buffer (RPMI (Gibco) with 10% FBS). After 1 hour, 2 μM monensin (Biolegend) and 3μg/ml Brefeldin A (BD) were added and incubated for 3 hours. Cells were stained with Live/Dead Fixable Aqua Stain (Thermo Fisher Scientific) and labelled with anti-CD56 (NCAM1) – Pe-Cy7 (clone: 5.1H11, Biolegend), anti-CD3 – APC-Cy7 (clone: sk7, BD), anti-CD14 – APC-Cy7 (clone: mΦp9, BD) and anti-CD19 – APC-Cy7 (clone: SJ25C1, BD). For intracellular cytokine staining, surface-stained cells were permeabilized using Cytofix/Cytoperm (BD) and cells were stained with anti-TNF-α - PE (clone: MAb11, BD) and anti-IFN-γ - APC (clone: B27, BD) antibodies. Cells were analyzed using Cytek Aurora (Cytek) and the data were analyzed using FlowJo Software (BD).
8
Flow Cytometry Analysis of Immune Cell Subsets
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Cryopreserved whole blood was thawed at room temperature, washed with RPMI 1640 plus 5mm EDTA (Life Technologies, Inc.), and diluted in RPMI 1640 supplemented with 10% FCS. Viability was determined using CD45 FITC (BD Biosciences Becton Dickinson). Cell pellets (100μl) were incubated with different panels of antibodies including anti-CD3-Alexa Fluor700, anti-CD4-APC, anti-CD127-PE-Cy5 (Bio Legend), anti-CD25-APC-Cy7, anti-CD14-APC-Cy7, anti-CD15-PE-Cy5, anti-CD19-PE-Cy7, anti-CD11c PE-Cy5, anti-HLA-DR-APC-Cy7, anti-CD123-PE-Cy7 (BD Biosciences) and anti-CD16-Pacific Orange (Invitrogen). In those panels to determine the production of chemokines, we also include anti-CCL22-PE and anti-IL-10- Pacific Blue (BD Biosciences). After 20 minutes of staining, cells were fixed using a BD FACS lysing solution (BD Biosciences). Cells were analyzed on a Fortessa flow cytometer.
Tregs were analyzed gating on CD3+ CD4+ CD25+ CD127low and mDCs were analyzed gating on Lineage (CD3, CD14, CD20 FITC) negative HLA-DR+CD11c+CD123+
Tregs were analyzed gating on CD3+ CD4+ CD25+ CD127low and mDCs were analyzed gating on Lineage (CD3, CD14, CD20 FITC) negative HLA-DR+CD11c+CD123+
9
Multiparametric flow cytometric analysis of monocyte subsets
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Whole blood samples were collected before and after IVIg administration. Immediately after collection, blood sample was incubated at room temperature for 20 min with the indicated fluorescently tagged monoclonal antibodies. Following RBC lysis (RT, 15 min) using FACS Lysing solution (Becton Dickinson), cells were washed twice and analyzed on a FACS Canto II (Becton Dickinson) flow cytometer. For simultaneous surface and intracellular staining, cell surface antigen staining was performed first, and cells were later resuspended in Buffer Perm/Wash 1 × solution, treated with fixation and permeabilization solution (4 °C, 30 min in the dark) and subjected to intracellular staining. Monocyte subpopulations were phenotypically identified by a 8-color flow cytometry single platform assay using anti-CD14-APC Cy7, CD16-FITC, CX3CR1-PerCP Cy5, HLA-DR-BV510, CD86-PE, CCR5-BV421, CCR2-APC, and TNF-PE mAbs (BD, Becton- Dickinson Biosciences, Franklin Lakes, NJ).
10
Comprehensive Flow Cytometry Analysis of PBMC
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Flow cytometry analysis was performed prospectively using samples from a part of patients at treatment initiation and 6 months post-treatment. Peripheral blood mononuclear cells were separated using density gradient centrifugation with the Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) and cryopreserved in the CELLBANKER 1 (Nippon Zenyaku Kogyo, Fukushima, Japan). The antibodies used for staining peripheral blood mononuclear cells were as follows; anti-CD4-VioGreen (Miltenyi Biotec, Bergisch Gladbach, Germany); anti-CD3-Paci c Blue/ uorescein isothiocyanate (FITC), anti-CD8-Paci c Blue, anti-CD14-(APC)-Cy7, anti-CD20 allophycocyanin-cyanine 7 (APC-Cy7), anti-CD25 phycoerythrin (PE)-Cy5, anti-CD27-PE-Cy7, anti-CD38-PE-Cy5, anti-CD45RO-PE-Cy7, anti-CD56-PE/PE-Cy7, anti-CD80-FITC, anti-CD86-PE-Cy5, anti-CD127-FITC, anti-CD161-APC and anti-chemokine (C-X-C motif) receptor 3 (CXCR3)-PE (all from BD Biosciences, Franklin Lakes, NJ, USA); anti-CD16-Brilliant Violet 510 and anti-CCR6-Brilliant Violet 421 (both from BioLegend, San Diego, CA, USA); and anti-mouse immunoglobulin G isotypematched controls (VioGreen from Miltenyi Biotec, the others from BD Biosciences). The peripheral cell subsets identi ed have been described in Supplementary table 1.
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