ASCs derived from sSAT, sRC and dSAT microenvironments were seeded in a 48-well plate. At 3 weeks of adipogenic induction, the culture medium was changed and after 24 h of culture medium change, the supernatant of all samples was collected and frozen at − 80 °C. The determination of proteins in the supernatant was carried out using the Cytometric Bead Array (CBA) technology (BD™) for recognition of human vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) and Chemokine (C–C motif) ligand 5 (CCL5) according to the manufacturer’s instructions. The data were acquired in FACSAria III (BD Biosciences) and analyzed using software FACSDiva 8.0 and FCPA Array (3.0) (BD Biosciences). Five replicates for two independent experiments (n = 2) were analyzed from three donors (n = 3).
Cytometric bead array technology
Manufactured by BD
Sourced in United States
The Cytometric Bead Array (CBA) technology is a multianalyte detection system. It allows for the simultaneous quantification of multiple proteins in a single sample. The technology utilizes color-coded beads that are coated with specific capture antibodies. These beads can then be used to detect and measure the concentration of target analytes in a sample.
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Lab products found in correlation
Facsdiva 8, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Lps from e coli k12, by InvivoGen (1 mentions)
Cpga odn 2336, by InvivoGen (1 mentions)
Facsverse, by BD (1 mentions)
Cobas 8000 system, by Roche (1 mentions)
7600 210 automatic analyser, by Hitachi (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
5 n ethylcarboxamidoadenosine neca, by Merck Group (1 mentions)
Glutamax, by Miltenyi Biotec (1 mentions)
Texmacs, by Miltenyi Biotec (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
5 protocols using cytometric bead array technology
1
Adipogenic Secretome Profiling
2
Dendritic Cell Maturation in Neuroblastoma Coculture
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We analyzed the maturation of DC upon TLR triggering in a two-dimensional coculture system, with cultures performed in the presence or not of neuroblastoma cell lines (50% confluent cells). Frozen PBMC from healthy donors were thawed, and cultured at 106 cells/mL, in 24-well culture plates, for 24 hours with or without TLR ligands [R848 (1 μg/mL), CpGA ODN-2336 (1.5 μmol/L) or LPS (from E. coli K12, 0.1 μg/mL) from Invivogen]. Supernatants were then harvested and frozen. IL12p70, IFNα2, TNFα, IL6, and IL8 concentrations were measured by Cytometric Bead Array (CBA) Technology (BD Biosciences).
3
Assessing cytokine secretion by myeloid cells
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Myeloid cells (granulocytes, mo/mΦ, and moDCs)-derived from 7-day BM cultures were purified by flow sorting. Then cells were cultured at 5 × 104 in 200 μL RPMI1640 supplemented with 10% FBS in U-bottom 96-well plates in the absence or the presence of LPS (1 μg/mL) or CpG (1 μM) for 20 h. For cytokine detection from the supernatants of in vitro assays, the indicated cytokines were detected using Cytometric Bead Array (CBA) technology (BD Biosciences) according to the manufacturer's instructions using a FACS Verse (BD Biosciences) cytometer.
4
Comprehensive Immunological Profiling Protocol
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Blood parameters, such as lymphocyte, neutrophil, and erythrocyte counts, were determined using a Sysmex XN-2000 haematology analyser (Sysmex, Tokyo, Japan). Liver, renal and cardiac function indicators such as alanine aminotransferase (ALT), aspartate transaminase (AST), creatinine, uric acid and lactate dehydrogenase were assayed using a Hitachi 7600-210 automatic analyser (Hitachi, Tokyo, Japan). Indexes of infection, C-reactive protein (CRP) and procalcitonin were detected using a Roche Cobas8000 system (Roche, Basel, Switzerland). In addition, cytokines, including IL-2, IL-4, IL-6, IL-10, and TNF-α, were measured via flow cytometry using Cytometric Bead Array (CBA) technology (BD Biosciences, San Jose, USA) [14 (link)].
5
Expanded NK Cell Cytokine Secretion
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FACS Aria-sorted NK cells from the spleen (Lin neg, 7AAD neg , CD49a neg , NKp46 + , NK1.1 + , CD49b + ) of the indicated mouse genotypes, were expanded for up to 10 days in complete RPMI 1640 containing 10% FBS, 1 µg/ml of anti-TGF-b1,2,3 blocking antibody (clone 1D11.16.8 from Bio X Cell, West Lebanon, NH), b-Me, GlutaMax and sodium pyruvate (Gibco). For cytokine secretion assays, cells were stimulated in the indicated concentrations of rTGF-b1, rActivin-A, or rIL-15 culture conditions in animal free / TGF-b1 free TexMACS medium (Miltenyi Biotec) containing b-Me, GlutaMax and sodium pyruvate for 48 hours. In certain proliferation assays, the pan adenosine receptor agonist 5'-(N-Ethylcarboxamido) adenosine (NECA) (Sigma Aldrich, St Louis. MO) was used at 1 µM in the culture media for comparison with TGF-b1. For the detection of cytokines in the supernatants of in vitro assays, IFN-g was measured by ELISA with the respective human or murine IFN-g Duoset Kit (R&D Systems) according to the manufacturer's instructions, while all other indicated cytokines were detected using Cytometric Bead Array (CBA) technology (BD Biosciences) according to the manufacturer's instructions.
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