All procedures involving human subjects were approved by the Institutional Review Boards of Yale University (IRB# 1005006865), and we complied with all relevant ethical regulations. Written informed consent was obtained from all participants prior to inclusion in the study. Human peripheral blood mononuclear cells were isolated by Ficoll (GE Healthcare) density gradient centrifugation and then stained with PE anti-CD3 (1:100, BioLegend, 300308), PE-Cy7 anti-CD19 (1:100, BD Pharmingen, 115520) and FITC anti-CD14 (1:100, BioLegend, 325604). Human CD14+CD3−CD19− monocytes were sorted in a BD FACSAria ΙΙ cell sorter.
Anti cd14 fitc
Manufactured by BioLegend
Sourced in United States
Anti-CD14-FITC is a fluorescently-labeled monoclonal antibody that binds to the CD14 surface antigen. CD14 is a glycosylphosphatidylinositol-anchored protein expressed on the surface of monocytes, macrophages, and neutrophils. The FITC (fluorescein isothiocyanate) label allows for the detection and analysis of CD14-positive cells using flow cytometry or other fluorescence-based methods.
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Lab products found in correlation
Anti cd3 pe, by BioLegend (2 mentions)
Anti cd3 fitc, by BioLegend (2 mentions)
Anti cd45 fitc, by BioLegend (2 mentions)
Anti cd73 pe, by BioLegend (2 mentions)
Anti cd105 pe, by BioLegend (2 mentions)
Anti nkp46 pe cy7, by BioLegend (2 mentions)
Anti cd19 apc cy7, by BioLegend (2 mentions)
Anti cd3 percp cy5, by Thermo Fisher Scientific (2 mentions)
Anti ulbp4 mab clone 709116, by R&D Systems (2 mentions)
Streptavidin bv421 sa bv421, by BioLegend (2 mentions)
Goat anti mouse igg pe gam pe, by BioLegend (2 mentions)
Fixable viability dye fvd efluor506, by Thermo Fisher Scientific (2 mentions)
Anti cd19 pe cy7, by BD (1 mentions)
Facsaria ιι cell sorter, by BD (1 mentions)
Ficoll, by GE Healthcare (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Anti cd40 pe, by BioLegend (1 mentions)
Fitc anti cd8α, by BioLegend (1 mentions)
Anti ifn γ apc, by BioLegend (1 mentions)
Anti cd206 pe, by BD (1 mentions)
13 protocols using anti cd14 fitc
1
Isolation and Sorting of Human Monocytes
2
Flow Cytometry Immunophenotyping Protocol
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The human monoclonal antibodies (mAbs) used for flow cytometry in this article included FITC-anti-CD3, FITC-anti-CD80, FITC-anti-CD163, FITC-anti-CD8α, FITC-anti-CD14, PE-anti-CD40, APC-anti-IFN-γ (all from BioLegend), BB515-anti-TLR2, and PE-anti-CD206 (all from BD Biosciences). Cells were scrapped from culture plates and incubated with human AB serum to saturate non-specific mAb binding before staining with the indicated fluorophore-labeled mAbs. For intracellular IFN-γ staining, cells were stimulated with Cell Activation Cocktail (with Brefeldin A; BioLegend) for 6 h according to the manufacturer's protocol. All data were collected using Novocyte flow cytometer (ACEA) and analyzed with FlowJo software.
3
Monocyte-Derived Dendritic Cell Maturation
4
Immunophenotypic Characterization of MSCs
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Isolated and expanded MSCs were observed for plastic adherence and fibroblast-like spindle morphology. For flow cytometric characterization, 100 μl of MSC resuspended in culture medium at a concentration of 1–1,5 × 106 MSC/ml was incubated with 1,5 μl anti-CD73 [PE] (BioLegend cat. no. 344044), 1,5 μl anti-CD90 [APC-Cy7] (BioLegend cat. no. 328132), 5 μl anti-CD105+ [APC] (BioLegend cat. no. 323208), 0,5 μl anti-CD14 [FITC] (BioLegend cat. no. 301804), 0,5 μl anti-CD19 [FITC] (BioLegend cat. no. 302206) 0,5 μl anti-CD31 [FITC] (BioLegend cat. no. 303104), 1,5 μl anti-CD45 [FITC] (BioLegend cat. no. 368508), 0,5 μl anti-HLA-DR [FITC] (BioLegend cat. no. 307604) and 1 μl of 1 mg/mL 7-Aminoactinomycin D (7-AAD, AAT Bioquest, cat. no. ABD-17501) for 30 min at room temperature in dark. After incubation, 3 mL of PBS with 0.1% BSA was added to the cells, followed by centrifugation at 500 g for 5 min. The pelleted cells were resuspended in 400 μl of PBS with 0.1% BSA, followed by analysis on a Novocyte 3000 (Agilent). The acquisition rate was kept below 1.000 for all analyses. A fluorescence minus one-guided gating strategy was applied to identify positive and negative events. In all applied MSC batches, ≥90% of the viable cells expressed the positive markers CD73, CD90, and CD105, while ≤2% expressed the negative markers CD14, CD19, CD31, CD45, and HLA-DR.
5
Multiparameter Flow Cytometry Analysis
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These following antibodies (Abs) were used for surface marker staining and ICS combined with flow cytometry (all Abs were from Biolegend): anti-CD3-PErCP-cy5.5 (UCHT1), anti-CD3-FITC (UCHT1), anti-CD3-PE (HIT3a), anti-CD4-APC (OKT4), anti-CD4-APC-cy7 (RPA-T4), anti-CD8-PE (RPA-T8), anti-CD8-APC-cy7 (SK1), anti-CD14-FITC (HCD14), anti-IFN-γ-PE (4S.B3), anti-TNF-α-APC (MAb11), anti-IL-17A-PE-cy7 (BL168), anti-Foxp3-PE (206D), anti-TGF-β-PE-cy7 (TW4-2F8), anti-Perforin-PC (dG9), anti-granzyme A-PE (CB9),.
6
Comprehensive Immune Cell Profiling
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T-cell activation/exhaustion was analyzed by staining with anti-CD3-PerCP, anti-CD4-PacificBlue, anti-CD8-APC, anti-CD25-PE/Cy7 and anti-CD279-PE (PD-1); ILCs by staining with anti-Lineage-Cocktail-APC (anti-CD3/CD14/CD16/CD19/CD20/CD56) and anti-CD127-PE; NK-cell subsets by staining with anti-CD3-PerCP, anti-CD14-FITC, anti-CD19-PE/Cy7, anti-CD56-APC/Cy7, anti-CD16-PacificBlue (all BioLegend, Fell, Germany), anti-NKG2A-APC (clone 131411) and anti-NKG2C-PE (both R&D Systems, Abingdon, UK) [20 (link)]. Samples were acquired on a CyAn ADP Analyzer (Beckman Coulter, Nyon, Switzerland) and data analyzed with FlowJo software vX.0.7 (FlowJo, Ashland, Oregon, USA).
7
Characterizing MSC Surface Markers and Apoptosis
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To characterize the expression of surface markers, the MSCs were immune-stained and detected by flow cytometry analysis. Antibodies used in this detection are anti-CD73-PE, anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD34-FITC, anti-CD19-PE, anti-CD45-APC and anti-HLA-DR-PE (Biolegend, San Diego, CA, USA).
To assess the apoptotic rates, H9c2 cells were seeded in a 6-well plate at 30,000 cells/well for 24 h and then desired treatments were performed for 24 h. Cells were stained using an Annexin V/PI staining kit (Beyotime, Shanghai, China) following the manufacturer’s instructions and detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
To assess the apoptotic rates, H9c2 cells were seeded in a 6-well plate at 30,000 cells/well for 24 h and then desired treatments were performed for 24 h. Cells were stained using an Annexin V/PI staining kit (Beyotime, Shanghai, China) following the manufacturer’s instructions and detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
8
Multimodal Immune Cell Profiling
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In the same set of experiments using SEA stimulated PBMCs described above, the effects of NM21-1480 on viability of monocytes, APCs, CD4 + T cells and CD8 + T cells was investigated. Viability of CD4 + T cells, CD8 + T cells, monocytes, CD11c+/CD123+ APCs and CD11c+ /CD86+ APCs was assessed by flow cytometry using antibodies specific for each marker cited above, anti-CD8-FITC (BioLegend, 300906), anti-TCR ɑ/β-APC (BioLegend, 306718), anti-CD11c-PE/Cy7 (BioLegend, 344710), anti-CD123-PE (BioLegend, 306006), anti-CD14-FITC (BioLegend, 325604), anti-CD16-APC/Cy7 (BioLegend, 302018). Apoptotic cells were quantified using Annexin V-APC (BioLegend, 640941).
9
Antibody Panel for ULBP4 Detection
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Anti-ULBP4 mAb (clone 709116) was purchased from R&D (Minneapolis, MN, USA). The ULBP4-specific mAb DUMO1 was previously described [32 (link)]. Anti-CD3-PerCP-Cy5.5 and fixable viability dye(FVD)-eFluor506 were from eBioscience (SanDiego, CA, USA), anti-CD19-APC-Cy7, anti-CD14-FITC, anti-NKp46-PE-Cy7, streptavidin-BV421 (SA-BV421) and goat-anti-mouse-IgG-PE (GaM-PE) were from Biolegend (SanDiego, CA, USA).
10
Macrophage Differentiation of Transgenic U937 Cells
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To differentiate transgenic U937 cells to monocyte/macrophage, 1 × 106 cells/mL were incubated with complete RPMI supplemented with a final concentration of 10 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Gillingham, UK) in 24 well plates for 48 h. To control the metabolic and redox contribution of BCAT1, the media was supplemented with 20 mM Gabapentin (Sigma-Aldrich, Gillingham, UK) and/or 10 mM NAC respectively. Intracellular ROS ± PMA was measured by DCFDA staining as previously described. To examine cellular differentiation, U937 cells were incubated with anti-CD14 FITC (Biolegend), anti-CD68 PE (Biolegend), anti-CD36 PE (Biolegend, London, UK) and anti-CD11b FITC (Biolegend, London, UK) prior to flow cytometric analysis, where % positive cells and median fluorescence intensity (MFI) were evaluated. PMA treated cells were stained with Giemsa and imaged via light microscopy to evaluate macrophage morphology.
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