Bone marrow cells isolated from C57BL/6, alox5−/−, S129, and lta4h−/− mice were applied to LIVE/DEAD fixable Dead Cell staining by incubating with Near-IR fluorescent reactive dye (Thermo Fisher Scientific) in HBSS at room temperature for 15min in the dark, followed by two washes with HBSS. The cells were then resuspended in eBioscienceTM Flow Cytometry staining buffer (Thermo Fisher Scientific) and incubated with rat anti-mouse CD16/CD32 monoclonal antibody on ice for 10 min to block the Fc receptor. The following antibodies were then added to stain the cells on ice for 15 to 30 min in the dark: PE-conjugated rat anti-mouse CD45 (BioLegend), PerCP-Cyanine5.5-conjugated rat anti-mouse CD11b (Thermo Fisher Scientific), APC-conjugated rat anti-mouse Ly-6G (Thermo Fisher Scientific), Alexa Fluor 488-conjugated rabbit anti-mouse BLT1 (Bioss Antibodies). The cells were washed and applied to flow cytometry analysis using Attune NxT Flow Cytometer (Thermo Fisher Scientific). The data were analyzed by FlowJo. Neutrophils were gated as CD45+CD11b+Ly-6G+ live cells and the mean fluorescence intensity (MFI) of BLT1 on neutrophils was compared between C57BL/6, alox5−/−, S129, and lta4h−/− genotypes.
Near ir fluorescent reactive dye
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Belgium, United States
The Near-IR fluorescent reactive dye is a specialized laboratory reagent designed for various research and analytical applications. Its core function is to provide a near-infrared fluorescent label that can be covalently attached to target molecules, enabling sensitive detection and analysis using appropriate instrumentation.
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Lab products found in correlation
Easysep mouse cd4 t cell isolation kit, by STEMCELL (2 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pe conjugated rat anti mouse cd45, by BioLegend (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Sambucus nigra, by Vector Laboratories (1 mentions)
Succinylated wheat germ agglutinin, by Vector Laboratories (1 mentions)
Lens culinaris agglutinin, by Vector Laboratories (1 mentions)
Griffonia simplicifolia, by Vector Laboratories (1 mentions)
Pisum sativum agglutinin, by Vector Laboratories (1 mentions)
Concanavalin a type 4, by Merck Group (1 mentions)
Phaseolus vulgaris erythroagglutinin, by Vector Laboratories (1 mentions)
Phaseolus vulgaris leucoagglutinin, by Vector Laboratories (1 mentions)
Streptavidin pe, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti ifn γ pe, by Thermo Fisher Scientific (1 mentions)
Canto 2 flow cytometry, by BD (1 mentions)
Anti il 4 pe, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Concanamycin a, by MedChemExpress (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
9 protocols using near ir fluorescent reactive dye
1
Neutrophil BLT1 Expression Across Genotypes
2
Glycan Profiling of Confluent Cells
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Confluent cells were detached from cell culture dishes by gentle resuspension in PBS and were transferred to V-bottom 96-well plates for staining (1–2 × 106 cells/well). Prior to this, control cells were treated with 4 µg/ml kifunensine (Bio-Techne, Minneapolis, MN, USA) overnight. Cells were washed with staining buffer (PBS supplemented with 2% (v/v) fetal calf serum, 2 mM EDTA) and were then stained successively with a near-IR fluorescent reactive dye (1:500, Thermo Fisher Scientific), biotin-conjugated lectins and streptavidin-PE (1:1000, Thermo Fisher Scientific) diluted in FACS buffer. The following biotinylated lectins were used: Sambucus nigra (5 µg/ml), Griffonia simplicifolia (0.5 µg/ml), Pisum sativum agglutinin (1 µg/ml), Lens culinaris agglutinin (0.5 µg/ml), Phaseolus vulgaris erythroagglutinin (1 µg/ml), Phaseolus vulgaris leucoagglutinin (1 µg/ml), and succinylated wheat germ agglutinin (2 µg/ml) (all from Vector laboratories, San Francisco, CA, USA) as well as Concanavalin A Type IV (1 µg/ml) and Tritium vulgaris (0.5 µg/ml) (from Sigma–Aldrich). Stained cells were fixed with 2% formaldehyde (FA) in PBS (Polysciences) and were analysed using a FACSCanto II and the FlowJo 10.6.2 software package (Becton Dickinson).
3
Characterization of iNKT Cell Phenotype
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PBMCs or expanded iNKT cells were harvested, washed with PBS, and stained with APC-PBS57-CD1d tetramer at 37°C in the dark for 20 minutes. Cells were then incubated with near-IR fluorescent reactive dye (Invitrogen, Darmstadt, Germany), PE-Cy7-anti-CD3 and PerCP-Cy5.5-anti-CD4 at 4°C for 20 minutes. After incubation, cells were extensively washed, fixed, and permeabilized using a BD Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA), according to the manufacturer's instructions. These cells were finally stained with either PE-anti-IFN-γ or PE-anti-IL-4 (eBioscience) and analyzed on a BD Canto II flow cytometry.
4
Evaluating Caspase-3 Activation in Ishikawa Cells
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Ishikawa cells were pre-treated with either JWG-071 or vehicle (0.05% v/v DMSO) for 12 h and detached. In parallel, eNK cells were pre-treated with 1 µM Concanamycin-A (MedChemExpress) or vehicle (0.1% v/v DMSO) for 2 h. eNK cells were co-cultured with Ishikawa EC cells at 1:5 (effector:target) ratio in the presence of 1 µM Concanamycin A or vehicle for 4 h in U-bottom 96-well plates (Corning), and active-caspase-3 in Ishikawa cells was analyzed by flow cytometry. Briefly, samples were stained with mAbs specific for CD45-BV510 (clone HI30) and EpCAM-AF647 (clone 9C4), together with Near IR-fluorescent reactive dye (Invitrogen) to exclude dead cells, permeabilized with wash/perm buffer, stained with anti-cleaved caspase-3-FITC antibody (BD Pharmingen, clone C92-605), acquired on a BD-LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (vX.0.7, TreeStar). Percentages of active Caspase 3+ cells were assessed in viable Ishikawa cells (EpCAM+/CD45-). Antibodies used in this process are listed in Supplementary Table 4 .
5
Isolation and Activation of Murine CD4+ T Cells
6
Megakaryocytic Differentiation Assay
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Megakaryocytic differentiation was monitored by flow cytometry as previously described30 (link) using fluorescein isothiocyanate (FITC)-, R-phycoerythrin (RPE)- or allophycocyanin (APC)-conjugated monoclonal antibodies against CD61 (clone Y2/51, Dako, Ely, UK) and/or CD42b (clone HIP1, BD Biosciences, Oxford, UK). Dead cells were excluded using 7-amino actinomycin D (1 μg/mL, Invitrogen, Paisley, UK) or near-IR fluorescent reactive dye (1:250 dilution, Invitrogen, Paisley, UK), as appropriate. In experiments where the relative antigen expression was estimated using the median fluorescent intensity, nonspecific fluorescence of cells stained with an isotype-matched control antibody was subtracted from all determinations. When indicated, the number of differentiated cells was estimated by multiplying the percentage of antigen-positive cells by the total number of live cells in the culture.
7
Isolation and Activation of CD4+ T Cells
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Both wild-type and IFNAR1 -/-mice were on a C57BL/6 background, with IFNAR1 -/-mice previously described in (REF). Splenic single cell suspensions were generated, and red blood cells lysed using ACK lysis buffer (Life Technologies). CD4 + T cells were isolated by negative selection using the Stem Cell Technologies EasySep Mouse CD4 + T Cell Isolation Kit (Tukwila, WA), resulting in a CD4 + purity of 95-98%, and a CD3 + purity of ~99%. CD4 + T cells were then plated at 180,000 cells per well in a 96-well plate and activated with plate-bound anti-CD3 (2 g/ml) and soluble anti-CD28 (1 g/ml) for the indicated time before harvesting and staining with near-IR fluorescent reactive dye (Invitrogen).
8
Evaluating Drug Cytotoxicity and Apoptosis
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To assess drug cytotoxicity, PBMCs were incubated with or without drugs in a 96-well round bottom plate (Thermo Fisher, Asse, Belgium) at 2 × 105/200 μL/well. After 2 days, cells were washed with PBS and stained with 0.1 μL near-IR fluorescent reactive dye (Life technologies, Ghent, Belgium) in 100 μL PBS for 20 min at room temperature in dark. Stained cells were measured by flow cytometry (FACSCanto, BD biosciences, Belgium).
To measure apoptosis, PBMCs were incubated with or without drugs in a 96-well round bottom plate at 2 × 105/200 μL/well. After 2 days, cells were washed with PBS and stained with 2.5 μL Annexin-V-APC (Biosciences, Aalst, Belgium) in 100 μL staining buffer containing 10 mmol/L HEPES pH7.4, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2 for 20 min at room temperature in dark. Cells were then washed with staining buffer and incubated with 100 μL 0.1 μg/mL propidium iodide (PI) (Thermo Fisher, Asse, Belgium) for 5 min at room temperature in dark. Stained cells were measured by flow cytometry (FACSCanto, BD, Aalst, Belgium).
To measure apoptosis, PBMCs were incubated with or without drugs in a 96-well round bottom plate at 2 × 105/200 μL/well. After 2 days, cells were washed with PBS and stained with 2.5 μL Annexin-V-APC (Biosciences, Aalst, Belgium) in 100 μL staining buffer containing 10 mmol/L HEPES pH7.4, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2 for 20 min at room temperature in dark. Cells were then washed with staining buffer and incubated with 100 μL 0.1 μg/mL propidium iodide (PI) (Thermo Fisher, Asse, Belgium) for 5 min at room temperature in dark. Stained cells were measured by flow cytometry (FACSCanto, BD, Aalst, Belgium).
9
Splenocyte Isolation and Flow Cytometry
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Spleens obtained from mice between the age of 12 and 35 weeks were smashed with a plunger against a 70 μm nylon cell strainer (BD Biosciences, CA, USA) while being flushed with 1 × phosphate buffered saline (PBS) + 2 mM EDTA to obtain single cell suspension.
Flow cytometry was performed using the BD Fortessa X-20 cell analyzer (BD Bioscience, CA, USA). Data were analyzed using the FlowJo data acquisition and analysis software. Fc receptor blocker was used during sample preparation. For intracellular staining, intracellular fixation and permeabilization kit (eBioscience, CA, USA) was used as per manufacturer's instructions. Antibodies used are listed inSupplementary Table 1 .
For intracellular IL-10 and IL-17 expression analysis, splenocytes were stimulated in vitro with anti-CD3 antibody (0.5 μg/ml, clone 145-2C11, BD Pharmingen, USA) for 48 h with addition for monensin (Biolegend, MN, USA) for the last 18 h. Flow cytometry in these experiments involved staining for live dead cells with the Near IR Fluorescent Reactive Dye (Life Technologies, CA, USA). Live cells were gated and analyzed.
Flow cytometry was performed using the BD Fortessa X-20 cell analyzer (BD Bioscience, CA, USA). Data were analyzed using the FlowJo data acquisition and analysis software. Fc receptor blocker was used during sample preparation. For intracellular staining, intracellular fixation and permeabilization kit (eBioscience, CA, USA) was used as per manufacturer's instructions. Antibodies used are listed in
For intracellular IL-10 and IL-17 expression analysis, splenocytes were stimulated in vitro with anti-CD3 antibody (0.5 μg/ml, clone 145-2C11, BD Pharmingen, USA) for 48 h with addition for monensin (Biolegend, MN, USA) for the last 18 h. Flow cytometry in these experiments involved staining for live dead cells with the Near IR Fluorescent Reactive Dye (Life Technologies, CA, USA). Live cells were gated and analyzed.
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