Cba flex set system

Manufactured by BD

Sourced in United States

The BD™ CBA Flex Set System is a multiplex assay system designed for the quantitative measurement of multiple analytes in a single sample. The system utilizes flow cytometry technology to analyze a panel of pre-defined capture beads, each coated with specific antibodies to detect various target proteins or cytokines.

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19 protocols using cba flex set system

Cytokine Profiling of Immune Cell Cultures

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Supernatants of DC, PS/NTreg and PS/NTeff cultures, and PP, PN, NP, NN cocultures were harvested to determine cytokine production. Secreted IL-12p70, IL-10 and TGF-β1 levels were measured with ELISA (R&D, USA) and BD™ CBA Flex Set system (BD Biosciences) according to the manufacturers’ instructions. IL-17a and IFN-γ were quantified with BD™ CBA Flex Set system (BD Biosciences) according to the manufacturer’s instructions.

Li M., Eckl J., Geiger C., Schendel D.J, & Pohla H. (2017). A novel and effective method to generate human porcine-specific regulatory T cells with high expression of IL-10, TGF-β1 and IL-35. Scientific Reports, 7, 3974.

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Quantifying Pro- and Anti-Inflammatory Cytokines

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The plasma pro- and anti- inflammatory cytokines were quantified by human cytometric bead array (CBA) kits (BD Biosciences Pharmingen, San Jose, CA, USA) for IL-1β (interleukin-1 beta), IL-6, IL-8, IL-10, IL-12p70 and TNF (tumour necrosis factor), and by CBA Flex Set system (BD Biosciences Pharmingen, San Jose, CA, USA) for IL-2, IL-4, IL-5, IL-13, IL-17A and IFN-γ (interferon gamma). Standard curves for each cytokine were generated by using the reference cytokine concentrations supplied with the kits. All standards and plasma samples were acquired on Accuri™ C6 Plus personal flow cytometer (BD Biosciences). Data analysis was performed using the FCAP Array software version 3.0.1 (BD/Softflow, Pécs, Hungary) and the values were expressed in pg/mL.

Maneerattanasak S., Gosi P., Krudsood S., Chimma P., Tongshoob J., Mahakunkijcharoen Y., Sukasem C., Imwong M., Snounou G, & Khusmith S. (2017). Molecular and immunological analyses of confirmed Plasmodium vivax relapse episodes. Malaria Journal, 16, 228.

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Treg-DC Coculture Cytokine Analysis

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Splenic MACS isolated CD4⁺CD25⁺ Treg (Miltenyi Biotec, 130091041) of CD18^Foxp3 mice and WT littermates, as well as MACS isolated DC (Miltenyi Biotec, 130100875) from WT mice were cocultured (3:1; DC/Treg) in RPMI from Thermo Fisher Scientific (supplemented with 10% FCS [PAN Biotech], 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μM β-mercaptoethanol, 50 μM natrium pyruvate; Thermo Fisher Scientific) for 48 hours (5% CO₂, 37°C). Cytokine analysis of the supernatant was performed with CBA flex set system (BD Biosciences; IL-6, 519004153; IL-1β, 51-9005788) as described. Beads were detected with an Attune flow cytometer and analyzed with FCAP Array software.

Klaus T., Wilson A.S., Vicari E., Hadaschik E., Klein M., Helbich S.S., Kamenjarin N., Hodapp K., Schunke J., Haist M., Butsch F., Probst H.C., Enk A.H., Mahnke K., Waisman A., Bednarczyk M., Bros M., Bopp T, & Grabbe S. (2022). Impaired Treg-DC interactions contribute to autoimmunity in leukocyte adhesion deficiency type 1. JCI Insight, 7(24), e162580.

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Antibody-Mediated Cytokine Analysis

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Fluorochrome-conjugated antibodies for flow cytometry were purchased from BD Biosciences (Heidelberg, Germany), eBioscience (Frankfurt, Germany), and BioLegend (London, UK). Cytokine secretion was measured from supernatants using mouse CBA Flex Set System according to the manufacturer's protocol (BD Biosciences).

Chessa F., Mathow D., Wang S., Hielscher T., Atzberger A., Porubsky S., Gretz N., Burgdorf S., Gröne H.J, & Popovic Z.V. (2016). The renal microenvironment modifies dendritic cell phenotype. Kidney international, 89(1).

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Cytokine Release Assay Protocol

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For anti-CD3 antibody/anti-CD28 antibody-stimulated cytokine release assay, PBMC suspensions (100 μl/well, 1 × 10⁵ cells/well) were seeded into anti-CD3 antibody (Thermo Fisher Scientific, Waltham, MA, USA) pre-coated 96-well tissue culture plates and incubated with test compounds for 30 min at 37°C. The cells were then stimulated with anti-CD28 antibody (0.5 μg/ml; Thermo Fisher Scientific) solution and incubated for 24 h at 37°C. For the IL-2–stimulated cytokine release assay, PBMC suspensions (100 μl/well, 1 × 10⁵ cells/well) were pre-incubated with the test compounds for 30 min at 37°C before stimulation with IL-2 (100 ng/ml) and incubation for 72 h at 37°C. Normal human dermal fibroblast cells (100 μl/well, 1 × 10⁴ cells/well) were seeded into flat-bottomed 96-well tissue culture plates 2 days before stimulation with IL-6 (100 ng/ml) or IFN-α (1000 U/ml) for 24 h and drug treatment. Culture supernatants were harvested after incubation, and cytokine concentrations were determined using a BD CBA Flex Set System (BD Biosciences). Cell viability was also measured using a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA).

Kitanaga Y., Imamura E., Nakahara Y., Fukahori H., Fujii Y., Kubo S., Nakayamada S, & Tanaka Y. (2019). In vitro pharmacological effects of peficitinib on lymphocyte activation: a potential treatment for systemic sclerosis with JAK inhibitors. Rheumatology (Oxford, England), 59(8), 1957-1968.

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Quantifying Inflammatory Cytokines in Sputum

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The concentration of the cytokines, IL-1β, IL-6, and IL-8, was analyzed in sputum supernatants using BD™ CBA Flex Set System (BD Bioscience-PharMingen, San Diego, CA, USA) according to the manufacturer's instructions [29 (link), 31 (link)]. DTT (0.025%) was added to the standard curve and enzyme immunoassay buffer [32 (link)]. The lower detection limits of the cytokines were 2.3, 1.2, and 1.6 pg/mL for IL-1β, IL-8, and IL-6, respectively.

Jerkic S.P., Bächle L., Duecker R.P., Gronau L., Chiocchetti A.G., Zielen S, & Schubert R. (2023). Association between Polyunsaturated Fatty Acid Profile and Bronchial Inflammation in Bronchiolitis Obliterans. Mediators of Inflammation, 2023, 3406399.

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Cytokine and Chemokine Quantification in Sputum

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Concentrations of different cytokines/chemokines were determined in sputum samples using the BD^™ CBA Flex Set System for the measurement of IL-1β and IL-8 (BD Bioscience-PharMingen, San Diego, CA, USA) levels. Each BD^™ CBA Flex Set contained one bead population with distinct fluorescence intensity, as well as the appropriate phycoerythrin (PE) detection reagent and standard. The tests were performed according to the manufacturer’s advice, and samples were run in duplicate [27 , 28 ]. For analyses of the cytokines/ chemokines, we added the same concentration of DTT (0.025%) as in the sputum supernatant to the standard curve and enzyme immunoassay buffer.

Eickmeier O., Fussbroich D., Mueller K., Serve F., Smaczny C., Zielen S, & Schubert R. (2017). Pro-resolving lipid mediator Resolvin D1 serves as a marker of lung disease in cystic fibrosis. PLoS ONE, 12(2), e0171249.

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Cytokine and Chemokine Profiling of PBMC-MSC Interactions

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Cytokine/chemokine levels were measured in supernatants collected from PBMC stimulated with CD3/CD28 mAbs. 1 × 10⁵ PBMC were stimulated with CD3/CD28 mAbs and cultured in 96 well plates in the absence or presence of PLX-PAD, PLX-R18, or hAMSC cells at a PBMC:MSC ratio of 1:1. The supernatant was collected after 6 days and stored at −80 °C. Supernatants from PLX-PAD, PLX-R18, or hAMSC cells cultured alone were all included as controls. Each supernatant was thawed right before use in cytokine/chemokine assays. A multiplex bead-based immunoassay (BD CBA Flex Set system from BD Biosciences) was used to determine the levels of human IFN-γ (catalog number 560111), TNFα (catalog number 560112), IL-4 (catalog number 558262), IL-5 (catalog number 557288), IL-13 (catalog number 558450), IL-10 (catalog number 558274), TGF-β1 (catalog number 560429), IL-17A (catalog number 560383), Granzyme-A (GrzA) (catalog number 560299), Granzyme-B (GrzB) (catalog number 560304), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES/CCL5) (catalog number 558324). Samples were processed, according to the manufacturer’s instructions, acquired using a FACSAria III (BD Biosciences) and analyzed using FCAP Array software (BD Biosciences).

Papait A., Vertua E., Magatti M., Ceccariglia S., De Munari S., Silini A.R., Sheleg M., Ofir R, & Parolini O. (2020). Mesenchymal Stromal Cells from Fetal and Maternal Placenta Possess Key Similarities and Differences: Potential Implications for Their Applications in Regenerative Medicine. Cells, 9(1), 127.

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Cytokine and Chemokine Profiling of Monocyte-Macrophage Differentiation

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Cytokine/chemokine levels were measured in supernatants collected from differentiated monocytes, in the absence or presence of hAMTCs, CM, CM – PG or CM–IL‐6 (as described in Section 2.6). Supernatants from hAMTCs cultured alone in the transwell, or in the presence of the factors used to induce macrophage differentiation, along with the CM, CM – PG and CM–IL‐6 used to treat monocytes, were all included as additional controls (see supporting information, Figure S1). Each supernatant was collected, stored at −80 °C and thawed just before use in cytokine/chemokine assays.
A multiplex bead‐based immunoassay (BD CBA Flex Set system from BD Biosciences) was used to determine the levels of human IL‐1α, IL‐1β, IL‐8, IL‐10, IL‐12p70, interferon‐inducible protein 10 (IP‐10/CXCL10), monocyte chemoattractant protein 1 (MCP‐1/CCL2), monokine induced by IFNγ (MIG/CXCL9), macrophage inflammatory protein (MIP)‐1α, MIP‐1β, the CC chemokine regulated upon activation, normal T cell expressed and secreted (RANTES/CCL5) and TNFα. Samples were processed according to the manufacturer's instructions, acquired using a FACSAria (BD Biosciences) and analysed using FCAP Array software (BD Biosciences).

Magatti M., Vertua E., De Munari S., Caro M., Caruso M., Silini A., Delgado M, & Parolini O. (2016). Human amnion favours tissue repair by inducing the M1‐to‐M2 switch and enhancing M2 macrophage features. Journal of Tissue Engineering and Regenerative Medicine, 11(10), 2895-2911.

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Cytokine Induction by Nucleic Acids

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Example 5

Fifty micrograms of cM362-140, cM362-139 or poly(I:C) was intraperitoneally administered to wild-type C57BL/6J mice (9 weeks old, female) or TLR3KO mice (9 weeks old, female). Three mice were used per group. Each of the nucleic acids was prepared as a solution in RNase-free water. At 1 hour, 3 hours, and 6 hours after nucleic acid administration, the blood was collected from the tail vein, and the serum levels of TNF-α, IL-6 and IL-10 were measured. BD CBA Flex Set system was used for the measurement.

The results are shown in FIG. 7. The left panels show poly(I:C) administration, the center panels show cM362-139 administration, and the right panels show cM362-140 administration. The upper panels show TNF-α production, the middle panels show IL-6 production, and the lower panels show IL-10 production. The data were expressed as the mean value±SE (n=3). As apparent from FIG. 7, the in vivo cytokine production levels induced by cM362-140 were markedly low as compared with those induced by poly(I:C). The results indicate that cM362-140 has no risk of causing adverse effects such as cytokine storm when administered to a living body and is highly safe.

US10105385B1. Adjuvant composition (2018-10-23). National University Corporation Hokkaido University [JP]. Inventors: Tsukasa Seya [JP], Misako Matsumoto [JP].

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