Levels of serum cytokines (IL-17A, IL-22, and IL-23) were quantified using FlowCytomix™ kits (Bender MedSystems, Vienna, Austria) according to the manufacturer’s instructions. Data were collected on a FACSCalibur and analyzed with FlowCytomix Pro software (Bender MedSystems). Standard curves were determined for each cytokine. The detection sensitivity was 2.5 pg/mL for the IL-17A kit, 43.3 pg/mL for the IL-22 kit, and 21.9 pg/mL for the IL-23 kit.
Flowcytomix pro software
Manufactured by Thermo Fisher Scientific
Sourced in United States
FlowCytomix Pro Software is a flow cytometry data analysis software developed by Thermo Fisher Scientific. The software provides tools for processing and analyzing data generated from flow cytometry experiments.
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Lab products found in correlation
Flowcytomix, by Thermo Fisher Scientific (4 mentions)
Mouse regulatory t cell staining kit, by Thermo Fisher Scientific (2 mentions)
13plex flowcytomix multiplex kit, by Thermo Fisher Scientific (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Facscalibur, by Thermo Fisher Scientific (2 mentions)
Microwin software, by Berthold Technologies (2 mentions)
Facscanto 2, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Lsrfortessa, by BD (1 mentions)
Human th1 th2 11plex flowcytomix kit, by Thermo Fisher Scientific (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Flow cytometry instrument, by BD (1 mentions)
Gallios 3 l 10c, by Beckman Coulter (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Softmax pro software, by Molecular Devices (1 mentions)
Opteia human ifn γ elisa set, by BD (1 mentions)
Opteia human ifn gamma elisa set, by BD (1 mentions)
Flowcytomix kit, by Thermo Fisher Scientific (1 mentions)
13plex flowcytomix kit, by Thermo Fisher Scientific (1 mentions)
24 protocols using flowcytomix pro software
1
Quantification of Serum Cytokines
2
Multiplex Biomarker Analysis in Obesity
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Leptin, resistin, soluble CD40 ligand (sCD40L), sICAM-1, soluble tumor necrosis factor receptor (sTNF-R), and monocyte chemoattractant protein 1 (MCP-1) were analysed using the eBioscience® FlowCytomix™ Human Obesity 9plex Kit (Bender MedSystems GmbH, Austria) following the manufacturer’s instructions. A standard protein dilution and human plasma samples were incubated with a Bead and Conjugate Mixture for two hours. After washing with assay buffer, a Streptavidin-PE Solution was added and incubated for one hour. Subsequently, samples and standard protein dilutions were washed twice, re-suspended in assay buffer, and analysed by flow cytometry using LSR Fortessa (BD Biosciences, San Diego, USA) with FlowCytomix Pro Software (Bender MedSystems GmbH, Vienna, Austria).
3
Cytokine Profiling of Lyme Disease
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PBMCs were co-incubated with B. burgdorferi for 20 hrs. Cell-free culture supernatants were prepared by removal of PBMCs at 300×g for 10 min followed by centrifugation of the supernatant at 9000×g for 10 min to remove non-adherent spirochetes and cellular debris. Cell-free supernatants were aliquoted and stored at −20°C until further use. Concentrations of human IFN-α and IFN–λ1 were quantitated using the Human Verikine IFN-α (PBL Biomedical Laboratories) or the Human IL-29 ELISA Ready-Set Go! Kit (eBioscience), respectively. Protein levels of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p70, TNF-α and TNF-β in cell-free culture supernatants were measured using the FlowCytomix Human Th1/Th2 11plex Kit (BMS810FF;Bender MedSystems) according to the manufacturer’s instructions. Data were acquired with a MACSQuant analyzer (Miltenyi Biotec) and analyzed using FlowCytomix Pro Software (Bender MedSystems).
4
Quantifying Cytokines in BALF
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A lethal dose of ketamine and lompun was injected to mice intraperitoneally to sacrifice. BALF was collected via insertion of silicon tube to the trachea. Mice were given 1.8 ml of PBS into lung and >1.5 ml of buffer was retrieved consistently. To detect cytokines, BALF was centrifuged at 1100×g for 10 min. Supernatants were obtained and stored at 4°C.
Interleukin-4, interferon gamma (IFN-γ), IL-17, and GM-CSF levels in BALF were measured according to the method of Chen et al.[25 (link)] Briefly, we added BALF or serially diluted cytokine standards in 96-well micro plate (Millipore, Billerica, MA, USA), antibody-linked fluorescent bead and secondary antibody biotin-conjugated were added, and then incubated at room temperature (RT) for 2 h. After washing, streptavidin-phycoerythrin was added, and samples were incubated at RT for 1 h. After washing, the samples were replaced in a 5-ml tube (SPL, Gyoungi-do, Korea), and 300 μl of assay buffer was added. Fluorescence from micro plate was detected using flow cytometry instrument (BD, Franklin Lakes, NJ, USA). Data were analyzed using FlowCytomix Pro software (Bender Medsystems, Vienna, Austria) and expressed as picogram per milliliter.
Interleukin-4, interferon gamma (IFN-γ), IL-17, and GM-CSF levels in BALF were measured according to the method of Chen et al.[25 (link)] Briefly, we added BALF or serially diluted cytokine standards in 96-well micro plate (Millipore, Billerica, MA, USA), antibody-linked fluorescent bead and secondary antibody biotin-conjugated were added, and then incubated at room temperature (RT) for 2 h. After washing, streptavidin-phycoerythrin was added, and samples were incubated at RT for 1 h. After washing, the samples were replaced in a 5-ml tube (SPL, Gyoungi-do, Korea), and 300 μl of assay buffer was added. Fluorescence from micro plate was detected using flow cytometry instrument (BD, Franklin Lakes, NJ, USA). Data were analyzed using FlowCytomix Pro software (Bender Medsystems, Vienna, Austria) and expressed as picogram per milliliter.
5
Cytokine Profiling of PBMC-MSC Cocultures
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Cytokine analysis in coculture supernatants was performed by seeding 105 PBMC in 96 flat-bottom well plates (TPP, Trasadingen, Switzerland), with and without the different MSC clones at 1 : 10 ratio (MSC : PBMC), in complete RPMI medium for 96 hours. They were stimulated with 10 μg/mL of PHA, and no stimulation condition was used as control. Supernatants were then collected, centrifuged at 360g, and frozen at −20°C until analysis. Cytokine detection was performed by flow cytometry (FacsCanto II, Becton Dickinson, San Diego, CA, USA) and the Human Th1/Th2/Th9/Th17/Th22 13plex FlowCytomix Multiplex kit from eBioscience (San Diego, CA, USA). The analysed cytokines were interleukin- (IL-) 1β, tumour necrosis factor- (TNF-) α, IL-10, interferon- (IFN-) γ, IL-4, IL-2, IL-22, IL-13, IL-17A, IL-9, IL-5, and IL-12p70. Data were analysed using FlowCytomix Pro Software (eBioscience, San Diego, CA, USA).
6
Cytokine Quantification in MLC Experiments
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The supernatant from MLC experiments was collected 2 and 6 days after culturing. Quantification of secreted interferon (IFN)‐γ, interleukin (IL)‐1‐β, IL‐2, IL‐5, IL‐9, IL‐10, IL‐13, IL‐22, sIL‐2R, tumour necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β was evalua‐ted by using a multiple cytometric bead array system FlowCytoMix (eBiosciences), according to the manufacturer's instructions. Samples were acquired with a FACSAria (BD Biosciences) and analysed with FlowCytoMix Pro software (eBiosciences).
7
Cytokine Measurement Protocol
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Cells from different setups were harvested at different time points (see figures) and centrifuged for 10 min at 1000×g. Supernatants were collected and used for measurement of cytokines either by bead-based ELISA or standard ELISA kits. For several cytokines, bead-based ELISA kits were used: Diacone Diaplex Th1/Th2/inflammation, Nordic Biosite, 880 100 010. For IL-6 only: Diacone Diaplex, Nordic Biosite, 880 030 001. Both bead-based assays were run on the Accuci C6 flow cytometer, and data were analyzed using the Flowcytomix Pro software (eBioscience).
8
Cytokine Profiling of Stimulated PBMCs
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Cytokine levels were determined in supernatants collected from the PBMC proliferation assays (anti-CD3 + anti-CD28 stimulated cells) after 6 days. All cytokine measurements were carried out with the human Th1/Th2/Th9/Th17/Th22 13plex bead array Kit (eBioscience, BMS817FF) according to the manufacturer's instructions. Briefly, samples were incubated with a bead mixture and biotin-conjugated cytokine specific antibody mixture light protected for 2 hours at room temperature. Samples were washed twice by adding assay buffer and spinning down with 200 g for 5 min. Staining with Streptavidin-PE solution was carried out under light protected conditions for 1 hour at room temperature, followed by 2 washing steps. Cytokines were assessed by flow cytometry (Gallios 3 L 10C, Beckman Coulter) and analyzed with FlowCytomixPro software (eBioscience).
9
Flow Cytometry Analysis of Cytokines
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Culture supernatants were analyzed by flow cytometry using Mouse Th1/Th2/Th17/Th22 13plex Kit FlowCytomix (eBioscience) according to the manufacturer’s instructions. The samples were acquired in LSR II flow cytometer. Analysis of data and quantification of cytokines was performed using the FlowCytomix Pro Software (eBioscience) on the basis of corresponding standards curves.
10
Quantification of Regulatory T-cells and Cytokines
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