The levels of the proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor α (TNF-α); that of the anti-inflammatory cytokine IL-10; and those of the chemokines interferon gamma-induced protein 10 (IP-10), IL-8, regulated on activation, normal T cell expressed and secreted chemokines (RANTES), monocyte chemotactic protein 1 (MCP-1), and monokine induced by IFN-γ (MIG), in both plasma and CSF, were determined using a cytometric bead array (BD Biosciences, San Diego, CA, USA) employing flow cytometry in accordance with the manufacturer’s protocol. The results are presented as means of data derived from duplicate tests. Data were analyzed using the BD cytometric bead array (software version 1.4). Theoretical limits of detection are shown in parentheses: IL-1β (2.3 pg/ml), IL-6 (1.6 pg/ml), TNF-α (1.2 pg/ml), IL-10 (4.7 pg/ml), IP-10 (0.5 pg/ml), IL-8 (1.2 pg/ml), RANTES (0.002 pg/ml), MCP-1 (1.3 pg/ml), and MIG (1.1 pg/ml).
Cytometric bead array
Manufactured by BD
Sourced in United States, Germany, United Kingdom, Australia, Sweden, Belgium, France
Cytometric bead array is a multiplex assay system designed for the quantitative measurement of multiple analytes in a single sample. It utilizes fluorescently-labeled beads to capture and detect target analytes, enabling the simultaneous analysis of multiple parameters.
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Lab products found in correlation
Fcap array software, by BD (22 mentions)
Facscanto 2 flow cytometer, by BD (17 mentions)
Facscanto 2, by BD (11 mentions)
Facscalibur, by BD (9 mentions)
Fcap array v3, by BD (8 mentions)
Facscalibur flow cytometer, by BD (8 mentions)
Lipopolysaccharides (lps), by Merck Group (7 mentions)
Lsrfortessa flow cytometer, by BD (7 mentions)
Cba flex set, by BD (6 mentions)
Ionomycin, by Merck Group (6 mentions)
Facsdiva software, by BD (6 mentions)
Lsrfortessa, by BD (5 mentions)
Facsverse flow cytometer, by BD (5 mentions)
Human il 2 enhanced sensitivity flex set, by BD (4 mentions)
Il 1β, by R&D Systems (4 mentions)
Fcap array software version 3, by BD (4 mentions)
Mouse th1 th2 th17 cytokine kit, by BD (4 mentions)
Facscanto, by BD (4 mentions)
Lsr 2 flow cytometer, by BD (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
430 protocols using cytometric bead array
1
Cytokine and Chemokine Profiling in Plasma and CSF
2
Quantifying Cytokine Responses to SARS-CoV-2 Proteins
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Mice were euthanized 120 days after the first immunization, and splenocytes were isolated. Splenocytes were cultured at 2 × 105 density in 200 µL RPMI medium (5% FBS, 2 mM L-glutamine, 1X NEAA, 1X vitamins, 1X antibiotic/antimitotic) and stimulated with recombinant S1, N, and RBD proteins (2 μg/mL) for 48 h at 37 °C. Supernatants were collected, centrifuged at 200× g, and stored at −20 °C until evaluation. The cytokines TNF-α, IFN-γ, IL-2, IL-4, and IL-5 were quantified according to the instructions of the Cytometric Bead Array (CBA) Mouse Th1/Th2 Cytokine using the Cytometric Bead Array (CBA) Mouse Th1/Th2 Cytokine (551287, BD Biosciences, Franklin, NJ, USA) kit. Data were collected using a Cytometer BD FACS Canto II.
3
Cytokine Profiling in Murine Schistosomiasis
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Cytokine assays were assessed in whole blood (without stimulus), from six mice per group per time point derived from cross-sectional assay➀, as previously described (35 (link)). Spleens were removed aseptically and processed as previously described (36 (link)). Cell viability counts were performed after staining with Trypan blue. Cells (1x106 per well) were plated on 24-well plates and stimulated with schistosomula extract (15 µg/ml) or concanavalin A (5 µg/ml, Sigma) as a positive control for cell reactivity, or saline as a negative control. After 72 h, culture supernatants were collected for cytokine assay using a Mouse Th1/Th2/Th17 CBA kit (BD™ Cytometric Bead Array). All procedures were conducted according to the manufacturer’s recommendations. Data was plotted by subtracting the baseline levels of cytokine production (no stimulus) for each group, and then displayed as a heatmap (z-score).
4
Purification and Activation of Murine Tregs
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Pooled cells from LNs and spleens were depleted of B cells and accessary cells by panning and T cells were enriched. The cells were stained with anti-CD8 and anti-B220 Abs, and CD4+ Treg cells were sorted on the basis of GFP (YFP) expression alone in CD8-negative population. The sorted cells were divided among 8 wells of a 96-well V-bottomed plate (2 × 105 cells/well) in 25 μl RPMI medium (10% FCS) with or without increasing doses of recombinant human IL-2 (0.016 to 12 U/ml), followed by incubation for 2 h at 37 °C. Depletion of IL-2 from the medium was assessed with the BD Cytometric Bead Array and Human IL-2 Enhanced Sensitivity Flex Set according to the manufacturer's instructions (BD Biosciences).
5
Comprehensive Lung Inflammation Analysis
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To perform bronchoalveolar lavage (BAL), a 20-gauge angiocath ligated into the trachea and 1 ml of sterile PBS was instilled, then removed through the angiocath; the process was repeated three times. The samples were pooled and a 200 μl aliquot of the BAL fluid was placed in a cytospin and centrifuged at 500 g for 10 min. The pellet was re-suspended in 1 ml of PBS, plated on glass slides, stained with crystal violet and subjected to a blinded manual cell count and differential. Cytokine and chemokine levels in the supernatant were measured using the BD Cytometric Bead Array (BD Biosciences). Samples were analysed in triplicate using the Mouse Inflammation Kit (BD Biosciences) according to the instructions provided. This kit detects interleukin (IL)-6, MCP-1 and tumour necrosis factor (TNF)-α. Total transforming growth factor (TGF)-β1 was measured using the TGF-β1 RII DuoSet enzyme-linked immunosorbent assay (ELISA, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Total interleukin (IL)-1β was measured using the IL-β1 Ready-Set-GO ELISA (Ebioscience, San Diego, CA) according to the manufacturer’s instructions. Caspase-1 activity was measured using the Caspase-1 Fluorometric Assay (R&D Systems), according to the manufacturer’s instructions.
6
Multiplex Analysis of Inflammatory Mediators
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Cytokine/chemokine levels in cell-free supernatants recovered from dorsal pouch exudates were measured using a multiplexed bead-based immunoassay (BD™ Cytometric Bead Array) according to the manufacturer's protocol. TNF (C8), IL-6 (B4), IL-10 (C4), IL-1β (E5), GM-CSF (B9), CCL2/MCP-1 (B7), CCL3/MIP-1ɑ (C7), CCL4/MIP-1β (C9) and CXCL1/KC (A9) levels were determined using a FACS Canto II flow cytometer with FCAP Array software, version 3.0 (BD Biosciences). CXCL2-3/MIP-2 measurements were per-formed using a commercially available ELISA kit (R&D systems Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. Plasma cytokines and G-CSF were measured using Eve Technologies (Calgary, AB, Canada).
7
Multiplex Cytokine Quantification
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Cell-free culture supernatants were analysed using a multiplex assay (BD Cytometric Bead Array) according to the manufacturer’s instructions to determine levels of IL-6 and TNF in biopsy supernatants.
8
Purification and Activation of Murine Tregs
9
Cytokine Profiling of Splenic Cells
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Cytokines levels in the splenic cell culture supernatant were measured by flow cytometry using BD cytometric bead array (CBA, BD Biosciences, Catalog No. 552364, Sweden) mouse soluble protein master buffer kit (IL-1α, GM-CSF, and TNF-α) according to the manufacturer’s instruction. Briefly, 1 × 106 spleen cells were collected from immunized mice, which were re-stimulated with MOG79-96 peptides (50 μg/mL) for 24 h at 37 °C.
10
Cytokine Profiling of T Cell-Macrophage Cocultures
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PBMC from BCG-vaccinated or LTBI individuals were stimulated with BCG or rested in medium for 7 days. On day 7, cells were washed, pelleted, counted, and they were added to wells of 96-well plates containing MAV or MAB-infected autologous macrophages at an effector to target (E:T) ratio of 10:1. On day 10, cytokines in cocultures were measured using cytokine Bead Array (CBA), a multiplex assay allowing simultaneous detection of IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ (BD Bioscience), was used to quantify levels of secreted cytokines in supernatants from co-cultures of MAV- and MAB-infected monocytes and T cells. The assays were performed according to the manufacturer's instructions and the data were analyzed using FCAP Array v 3.0 (BD) and a BD Cytometric Bead Array (BD Biosciences) software, in a FACS Canto TM flow cytometer (BD).
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