BALF was obtained as previously described (14 (link)). ELISA kits were used to measure serum leptin (Invitrogen, Carlsbad, CA), triacylglycerol (TAG, Pointe Scientific, Canton, MI), insulin (Uppsala, Sweden) and adiponectin (Abcam, Cambridge, UK). Blood glucose was measured via a 6h or 14h fast as described in figure legends. Further, BALF albumin was measured with the Mouse Albumin ELISA Kit (Genway Biotech, Inc., San Diego, CA). Total protein in BALF was measured via standard BCA assay (BCA kit, Sigma-Aldrich, St Louis, MO). BALF cytokines (IL-4, IFN-γ, MCP-1, RANTES, KC, IL-17A, IL-10 and TNF-α) were measured using a BioRad Bio-Plex assay (Hercules, CA) per manufacturer’s instructions. IL-4 was not measureable above the lower limit of the assay.
Bio plex assay
Manufactured by Bio-Rad
Sourced in United States, Italy, Germany
The Bio-Plex assay is a multiplex immunoassay system developed by Bio-Rad. It allows for the simultaneous detection and quantification of multiple analytes in a single sample. The system utilizes color-coded magnetic beads coated with specific capture antibodies to bind and measure targeted molecules. The core function of the Bio-Plex assay is to provide a platform for high-throughput, multiplexed analysis of various biomolecules, such as proteins, cytokines, and other biomarkers.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex 200 system, by Bio-Rad (5 mentions)
27 plex magnetic bead based immunoassay kit, by Bio-Rad (3 mentions)
Facscalibur, by BD (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
C57bl 6ncrlbr male mice, by Charles River Laboratories (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Vacutainer tube, by BD (2 mentions)
Bio plex manager 6, by Bio-Rad (2 mentions)
Bio plex manager software, by Bio-Rad (2 mentions)
Luminex xmap technology, by Bio-Rad (2 mentions)
Insulin, by Abcam (1 mentions)
Adiponectin, by Abcam (1 mentions)
Mouse albumin elisa kit, by Aviva Systems Biology (1 mentions)
Bca kit, by Merck Group (1 mentions)
Bio plex suspension array system, by Bio-Rad (1 mentions)
Anti cd80, by Thermo Fisher Scientific (1 mentions)
Anti cd86, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Elisa max standard set, by BioLegend (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
73 protocols using bio plex assay
1
Bronchoalveolar Lavage Fluid Analysis
2
Multiplex Cytokine Quantification Protocol
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Levels of IFN-γ and IL-10 were detected in plasma samples by a multiplex assay (Bio-Plex assay, Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions using the Luminex system (Luminex Corporation, Austin, TX, USA) and analysed with a Bio-Plex suspension array system (Bio-Rad Laboratories). Fluorescence intensity was transformed into cytokine concentration using the Bio-Plex manager software (version 3.0). A minimum of 100 beads per region were analysed. A curve fit was applied to each standard curve according to the manufacturer’s manual and sample concentrations were interpolated from the standard curves. The limit of detection was 0.79 pg/mL for IFN-γ and 2 pg/mL for IL-10.
3
Plasma Inflammatory Mediator Analysis
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Blood was collected in 0.5 M EDTA tubes by cardiac puncture and centrifuged at 1,000 x g for 10 min. Plasma supernatant was used for the analysis of inflammatory mediators using a multiplex assay (Bio-plex assay; Bio-Rad, Hercules, CA, United States).
4
Sarcoidosis Macrophage Cytokine Profiling
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Macrophages from BALF of sarcoidosis patients (n = 6) were isolated by adhesion to plastic as described previously.17 (link) The cells were treated with OATD-01 at 1 µM or vehicle (0, 1% dimethyl sulfoxide [DMSO]) for 24h and cell supernatants were collected for multiplex analysis of cytokines and chemokines (Bio-plex assay, Bio-rad).
5
Exosome-Mediated Macrophage Activation
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M1 macrophages (3 × 105) were incubated with 7.5 × 109 SF-derived exosomes for 24 hours or stimulated with 150 UI/ml IFNγ (Peprotech) for 12 hours and then with 10 ng/ml LPS (Sigma-Aldrich) for 24 hours. The stimulation with IFNγ/LPS was used as a positive control for these experiments. The expression level of the costimulatory molecules CD80 and CD86 was evaluated by flow cytometry using the anti-CD80 (eBiosciences) and anti-CD86 (eBiosciences) antibodies. Flow cytometry was carried out on the FACSCalibur (Becton Dickson) and data analysed using Flowing software. Supernatants were also harvested, centrifuged for 10 minutes at 14,000g, and cytokine/chemokine and MMP concentrations were quantified with a magnetic bead-based multiplex assay (Bio-plex Assay, Bio-Rad Laboratories). To ensure that cytokines were not previously present in our exosome preparation as contaminants, their presence was investigated directly in SF-derived isolated exosomes by magnetic bead-based multiplex assay.
6
Cytokine Profiling in Mouse Organs
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For measurement of cytokines (IFN-ƴ, TNF-α, IL-10) in mouse organ homogenate supernatants, ELISA MAX Standard Sets (Biolegend) were used according to the manufacturer’s instructions.
The levels of TNF-α, IFN-ƴ, IL-2, IL-4, IL-10, IL-12p70, and IL-17A in culture supernatants from spleen and lung cells of infected mice were analyzed following culture of 106 cells/well in 24-well plates for 48 hours with stimulation with Brucella spp. lysate or media using a Bio-Plex assay, according to the manufacturer’s instructions (Bio-Rad).
The levels of TNF-α, IFN-ƴ, IL-2, IL-4, IL-10, IL-12p70, and IL-17A in culture supernatants from spleen and lung cells of infected mice were analyzed following culture of 106 cells/well in 24-well plates for 48 hours with stimulation with Brucella spp. lysate or media using a Bio-Plex assay, according to the manufacturer’s instructions (Bio-Rad).
7
Cytokine and Leptin Quantification
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Cytokine levels in mouse serum were determined using the LEGENDplex Mouse Inflammation Panel (BioLegend), human leptin levels in serum using a customized LEGENDplex Panel (BioLegend) and mouse leptin using a customized Bio-Plex assay (Bio-Rad). The assays were performed according to the manufactures instructions, LEGENDplex assays were analyzed at BD LSRFortessa flow cytometer (Becton Dickinson), Bio-Plex assays at Luminex LX200 (Bio-Rad).
8
Chronic Pseudomonas aeruginosa Lung Infection
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C57BL/6NCrlBR male mice (8–10 weeks, Charles River) were challenged by intratracheal (i.t.) administration with an average of 5.6 × 105 colony forming units (CFUs) of the wt and ∆lasB RP45 strains or 3 × 105 CFUs of the wt and ∆lasB RP73 strains embedded in agar-beads (Bragonzi et al., 2009; (link)
Facchini et al., 2014 (link); Cigana et al., 2016b (link)) or with sterile empty agar-beads as control. Body weight and health status were monitored daily. Lung CFUs were analyzed at Days 2 and 7 post-infection. As reported previously, recovery of ≥1,000 CFUs of P. aeruginosa from Day 7 lung cultures is considered evidence that a chronic infection has been established (Lore et al., 2016 (link), 2018 (link)). Cytokine/chemokine/growth factor levels were measured in the supernatant of lung homogenates by Bio-Plex Assay (Bio-Rad), as described in the online data supplement. Additional details in accordance with the ARRIVE guidelines (Kilkenny et al., 2010 (link)) are reported in the online data supplement.
Facchini et al., 2014 (link); Cigana et al., 2016b (link)) or with sterile empty agar-beads as control. Body weight and health status were monitored daily. Lung CFUs were analyzed at Days 2 and 7 post-infection. As reported previously, recovery of ≥1,000 CFUs of P. aeruginosa from Day 7 lung cultures is considered evidence that a chronic infection has been established (Lore et al., 2016 (link), 2018 (link)). Cytokine/chemokine/growth factor levels were measured in the supernatant of lung homogenates by Bio-Plex Assay (Bio-Rad), as described in the online data supplement. Additional details in accordance with the ARRIVE guidelines (Kilkenny et al., 2010 (link)) are reported in the online data supplement.
9
Exosome-Induced Cytokine and Chemokine Response
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PBMC were isolated using Ficoll Hypaque (GE Healthcare, USA). Monocyte isolations were done using EasySep human monocyte isolation kit without CD16 depletion as per the manufacturer’s instructions (Stemcell Technologies, Canada). The viability of cells after isolation was ≥96%, and the purity was >92%. Monocytes or A549 cells were left untreated or treated with 10 μg of exosomes for 24 h and supernatant was subjected to cytokine and chemokine measurements using a custom 8-plex Bioplex assay (BioRad, USA) containing eight targets, namely interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin- 9 (IL-9), Monocyte Chemoattractant Protein-1 (MCP-1), Macrophage Inflammatory Protein-1 alpha (MIP-1α), Macrophage Inflammatory Protein-1 beta (MIP-1β), IFN-gamma-inducible protein-10 (IP-10), and the chemokine CCL5/regulated on activation, normal T cell expressed and secreted (RANTES), as per manufacturer’s instructions.
10
Efficacy of Aerosolized Antibiotics in Acute and Chronic Pseudomonas Infection
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Immunocompetent C57BL/6NCrlBR male mice (8–10 weeks; Charles River Laboratories, Calco, Italy) were challenged with 1×106 colony-forming units (CFUs) of the planktonic PAO1 strain for acute infection or 5×105 CFUs of the MDR-RP73 strain embedded in agar beads for chronic infection by intratracheal administration [12 (link)–14 (link)]. Mice were treated with TOB, COL or vehicle (water) by local administration using a Penn-Century MicroSprayer® Aerosoliser (aero) or by i.n. or systemic s.c. administration (figure 1 ). Body weight was monitored daily. Lung CFUs and cell counts in the bronchoalveolar lavage fluid (BALF) were analysed as described previously [13 (link), 18 (link), 19 (link)]. Cytokine/chemokine levels were measured in the supernatant of lung homogenates by Bioplex Assay (Bio-Rad Laboratories, Segrate, Italy). Pharmacokinetic (PK) profiles of TOB and COL in the lungs and plasma of P. aeruginosa-infected mice were evaluated by high-performance liquid chromatography–tandem mass spectrometry. Additional details in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines [20 (link)] are reported in the supplementary material .
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