The quantification of HGF, VEGF-A, TGF-β1, IL-8, platelet-derived growth factor-AA (PDGF-AA) and FGF-2 in the conditioned medium was performed using a commercially available kit (FlowCytomix™; eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Samples were acquired on a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) and the results obtained using FlowCytomix Pro 3.0 Software (eBioscience).
Flowcytomix pro 3
Manufactured by Thermo Fisher Scientific
Sourced in United States
FlowCytomix Pro 3.0 Software is a flow cytometry data analysis tool developed by Thermo Fisher Scientific. The software provides advanced features for the analysis and management of flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (3 mentions)
Cyan adp 9 color flow cytometer, by Beckman Coulter (2 mentions)
Cxcl10, by BD (2 mentions)
Flowcytomix custom assay kits, by Thermo Fisher Scientific (2 mentions)
Flowcytomix, by Thermo Fisher Scientific (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Flowcytomix simplex kit, by Thermo Fisher Scientific (1 mentions)
Cell lab quanta sc mpl, by Beckman Coulter (1 mentions)
Human il 8 elisa ready set go 2nd generation kit, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Ifn γ, by BioLegend (1 mentions)
Tnf α, by BD (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Flowcytomix kit, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by Thermo Fisher Scientific (1 mentions)
Fc500 cytometer, by BD (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Flowcytomix mouse th1 th2 kit, by Thermo Fisher Scientific (1 mentions)
14 protocols using flowcytomix pro 3
1
Quantification of Growth Factors in Conditioned Medium
2
Multiplex Cytokine Profiling of T-helper Cells
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Cell culture supernatants were collected at 24, 48, and 72 h posttransfection and analyzed simultaneously for 13 different human T-helper (Th) cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ, and TNF-α) using a multiplex bead-based assay with a commercial kit (BioLegend, San Diego, CA, USA, Catalogue number: 740721). The procedure was performed according to the manufacturer's instructions. Briefly, antibody-coated beads for each cytokine, which could be differentiated by their sizes and fluorochrome intensities, were incubated with supernatants or standards. Then, the secondary antibody conjugated to biotin was added, followed by adding PE-conjugated streptavidin. Data were analyzed using flow cytometry and the FlowCytomix Pro-3.0 software (eBioscience).
3
Quantification of Soluble Biomarkers
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Concentrations of sOPN, sCD44-v6 and sVCAM-1 in samples were measured separately using a FlowCytomix Simplex Kit (eBioscience, Vienna, Austria). The kit consists of fluorescent microspheres with an emission wavelength at 700 nm (5 μm diameter for sOPN and sCD44-v6 analysis and 4 μm diameter for sVCAM-1 analysis). Microspheres are coated with specific antibodies raised against each of the analytes (sOPN, sCD44-v6 or sVCAM-1). They also contain a biotin-conjugated second antibody and streptavidin-phycoerythrin emitting at 575 nm. Samples were run on a Cell Lab Quanta™ SC-MPL (Beckman Coulter, Fullerton, United States). Electronic volume versus side scatter gating was employed to exclude any sample particles other than 5 μm (4 μm) microspheres. Samples were acquired by Cell Lab QuantaTM SC-MPL software (Beckman Coulter, Fullerton, United States) and analyzed using Flowcytomix™ Pro 3.0 software (eBioscience, Vienna, Austria). The lower limits of detection of sOPN, sCD44-v6 and sVCAM-1 were 0.432 ng/ml, 0.126 ng/ml and 0.9 ng/ml, respectively.
4
Multiplex Cytokine Profiling in Serum
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For cytokine detection in serum samples, a Th1/Th2/Th9/Th17/Th22 13plex Kit FlowCytomix (eBioscience, Frankfurt, Germany) was used. This bead-based analyte detection system measured levels of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p70, IL-13, IL-17A, IL-22 and TNF-α. Samples were measured using the FACS Canto II (BD Biosciences). Data were analyzed with FlowCytomix Pro 3.0 software (eBioscience). In addition, IL-8 levels in serum samples were determined using the Human IL-8 ELISA Ready-SET-Go! (2nd Generation) Kit from eBioscience according to the manufacturer’s instructions.
5
Cytokine and Chemokine Profiling of Cell Supernatants
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Supernatants from cultured cells were monitored using the human Th1/Th2/Th9/Th17/Th22 13-plex RTU FlowCytomix Kit (eBiosciences), and human chemokine 6-plex kit FlowCytomix (eBiosciences) according to the manufacturer’s instructions and acquired on a Cyan ADP 9-color flow cytometer (Beckman Coulter). Analyses were performed by FlowCytomix Pro 3.0 Software (eBiosciences). Some measurements were performed by ELISA with IFNγ (BioLegend), IL-9 (BioLegend), TNFα (BD Biosciences), CCL2 (BD Biosciences), CCL3 (R&D Systems), CCL4 (R&D Systems), CCL5 (R&D Systems), and CXCL10 (BD Biosciences) kits in accordance with the manufacturer’s recommendations.
6
Profiling Tumor-Derived Cytokine Landscape
7
PBMC Isolation, Cryopreservation and Cytokine Assay
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PBMCs isolation was performed as previously described [15] (link) and followed by cryo-preservation in liquid nitrogen until required [16] . PBMCs were thawed slowly (37°C) and then washed with RPMI 1640 medium supplemented with 10% FCS, gentamycin, penicillin/streptomycin (50 µg/ml) and L-glutamine (292.3 µg/ml), all from PAA (Linz, Austria). In 96-well plates, 1×105 PBMCs/well were left unstimulated or stimulated with OvAg (20 µg/ml) or αCD3/αCD28microbeads (40,000 beads/ml, Dynal/Invitrogen, Carlsbad, USA) in duplicate for 7 days. Cytokine levels were measured from pooled supernatants using a human FlowCytomix Multiplex Th1/Th2/Th9/Th17/Th22 13-plex kit (eBioscience, San Diego, CA, USA). Data were acquired on a FACS Canto flow cytometer (BD Biosciences, Heidelberg, Germany) and analyzed using FlowCytomix Pro3.0 software (eBioscience).
8
Cytokine Profiling in Immune Thrombocytopenia
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Peripheral blood was collected into heparin-anticoagulant vacutainer tubes before and 2 weeks after the treatment with HD-DXM [21 (link)]. Plasma was obtained from all subjects by centrifugation and stored at −80°C to determine the cytokines. Eight different cytokines were examined in the cITP patients and normal controls. The concentrations of IL-12p70, IL-23, IL-27, IFN-γ, IL-4 and IL-17A in the plasma were determined with specific FlowCytomix™ kits (eBioscience, San Diego, Calif., USA). The test samples were analyzed with a flow cytometer using a BD FACSAria instrument (Becton-Dickinson, San Jose, Calif., USA). The data for each cytokine were acquired using the FlowCytomix Pro 3.0 Software (eBioscience). For each analysis, up to 10,000 events were acquired. The minimum detectable concentrations of IL-12p70, IL-23, IL-27, IFN-γ, IL-4 and IL-17A were 1.5, 21.9, 10, 1.6, 20.8 and 2.5 pg/ml, respectively.
9
Multiplex Cytometric Bead Array for Cytokine Profiling
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Cytokine concentrations in sera or cell culture supernatants were determined using a multiplex cytometric bead array. The mouse Th1/Th2/Th17/Th22 13-plex FlowCytomix Multiplex kit (BMS822FF; eBioscience) was used according to the manufacturer’s instructions. In brief, samples or serial dilutions of cytokine standards were incubated with fluorescent cytokine capture beads and biotin-conjugated anticytokine Abs. Captured cytokines were detected by incubation with PE-conjugated streptavidin. Samples were analyzed on a flow cytometer (FACSCanto II), and data were processed using FlowCytomix Pro 3.0 software (eBioscience).
10
Quantifying Murine Cytokines via Flow Cytometry
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Murine cytokines were quantified by bead-based immunoassays adapted on flow cytometry, using the mouse Th1/Th2 10-plex FlowCytomixTM Kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions, using a filter plate and a vacuum filtration system for washing steps. Data were collected on a FC500® cytometer (BD Biosciences, San Jose, USA), and analyzed with FlowCytomix® Pro 3.0 Software (eBioscience). Standard curves were determined for each cytokine from a range of 27–20000 pg/ml. Serum concentrations were expressed in mean fluorescence intensity (MFI).
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