Eight wild-type and IFNAR−/− mice per group were infected with each strain as described above and the blood collected 12 h p.i. by intracardiac puncture following euthanasia and stabilization with EDTA (Sigma-Aldrich) as previously described (3 (link), 4 (link)). Plasma supernatants were collected following centrifugation at 10,000 × g for 10 min at 4°C and stored at −80°C. Plasmatic concentrations of TNF, IL-6, IL-12p70, CCL2, CCL3, and CXCL1 were measured using a custom-made cytokine Bio-Plex Pro™ assay (Bio-Rad) according to the manufacturer’s instructions. Acquisition was performed on the MAGPIX platform (Luminex®) and data analyzed using the Bio-Plex Manager 6.1 software (Bio-Rad).
Bio plex pro assay
Manufactured by Bio-Rad
Sourced in United States, Japan, France
The Bio-Plex Pro Assay is a multiplex immunoassay platform developed by Bio-Rad. The assay utilizes magnetic beads coated with specific capture antibodies to detect and quantify multiple analytes in a single sample. The platform combines bead-based technology with flow cytometry detection to provide simultaneous measurement of multiple targets in a high-throughput manner.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex manager 6, by Bio-Rad (7 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (7 mentions)
Bio plex 200 analyzer, by Bio-Rad (5 mentions)
Bio plex 200 system, by Bio-Rad (5 mentions)
Tissue extraction reagent 1, by Thermo Fisher Scientific (2 mentions)
Polytron pt 1200e system bundle, by Kinematica (2 mentions)
Procartaplex multiplex immunoassay, by Thermo Fisher Scientific (2 mentions)
Bio plex cell lysis kit, by Bio-Rad (2 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Applied biosystems 7900ht real time pcr system, by Thermo Fisher Scientific (1 mentions)
Il 12p40, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Il 12p35, by Thermo Fisher Scientific (1 mentions)
Irf 1, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Bio plex magpix system, by Bio-Rad (1 mentions)
Luminex 200 system, by Merck Group (1 mentions)
Human galectin 3 platinum elisa, by Thermo Fisher Scientific (1 mentions)
96 well u bottom plate, by Corning (1 mentions)
69 protocols using bio plex pro assay
1
Cytokine Quantification in Mouse Blood Post-Infection
2
Liver Protein Extraction and TNF-α Quantification
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For total protein extraction and TNF-α measurement, a modified protocol was applied as described [21 (link)]. In brief, liver tissue samples were homogenized with a QIAGEN Tissue Lyser II (Qiagen, Venlo, The Netherlands) for 2.5 min/25 Hz in extraction buffer containing 20 mM TrisHCl, 0.15 M NaCl, 2 mM EDTA, 1 mM EGTA, and a protease inhibitor cocktail (Sigma, Buchs, Switzerland). Samples were centrifuged at 1000× g for 10 min at 4 °C. Thereafter, the supernatants were removed and centrifuged a second time at 16,000× g for 120 min at 4 °C. The total protein concentration content was quantified with a coomassie dye-binding Bradford assay (Bio-Rad, Cressier, Switzerland). The TNF-α content was measured by a Luminex-based system using a mouse specific cytokines Bio-Plex Pro™ assay as described previously [21 (link)], following the standard protocol of the manufacturer (Bio-Rad).
3
Cytokine and HIV Replication Analysis
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Cytokines released in the supernatants were analyzed by using a customized Bio-Plex Pro Assay (BIO RAD) according to manufacturer’s instructions. The assay was able to evaluate the following cytokines and chemokines: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, G-CSF, IFN-γ, MCP-1, MIP-1b, RANTES, TNF-α, GM-CSF, IL-17.
HIV replication was evaluated by measuring p24 released in supernatants by a microelisa system (Vironostika HIV-1 antigen, Biomarieux) according to manufacturer’s instructions.
HIV replication was evaluated by measuring p24 released in supernatants by a microelisa system (Vironostika HIV-1 antigen, Biomarieux) according to manufacturer’s instructions.
4
Cytokine and Gene Expression Analysis of Stimulated Dendritic Cells
5
Cytokine Profiling of T Cell Assays
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Supernatant samples from T cell functional assays were retained following 20-h co-culture and frozen at −80°C until required for further use. Supernatants were subsequently analyzed for secreted human IL-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, GM-CSF, IFN-γ, MIP-1α, MIP-1β, and TNF-α according to the Bio-Plex Pro assay manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Plates were read using the Bio-Plex MAGPIX system (Bio-Rad, Hercules, CA, USA). Data were acquired using Bio-Plex Manager MP software and processed using Bio-Plex Data Pro Plus (both Bio-Rad, Hercules, CA, USA).
6
Bioactive Factors in Decellularized Organ Matrices
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Example 8
Growth factors retained in processed lung and liver tissue matrices were determined using a Bio-Plex Pro Assay (BioRad). Briefly, processed tissue was washed with saline and then freeze-dried and cryo-milled. Cryo-milled tissue (100 mg) was extracted in 1 ml tissue extraction reagent I (invitrogen) at 4° C. overnight. The supernatant was used for Bio-Plex Pro Assays (Human Angiogenesis Panel). The data shows that the decellularizing and processing of lung and liver tissues preserve the key growth factors (FGF, VEGF, PDGF) found in porcine organ matrices (table 4).
7
LPS-Induced Sickness Response in Mice
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Mice were i.p. injected with bacterial lipopolysaccharide (LPS) (0.20 mg/kg; serotype 0111:B4, Sigma-Aldrich) or sterile saline (0.9% NaCl, B. Braun Vet Care) [29 (link)]. Body temperature was measured 2, 4, 6 and 24 h post-injection to evaluate sickness. Three aged mice of each genotype were sacrificed between 6 and 24 h after LPS injection as they reached a humane endpoint. A second cohort of mice was sacrificed 3 h after LPS administration, using an overdose of pentobarbital (Dolethal®, 200 mg/kg i.p., Vetoquinol). Cardiac blood was collected in Li-heparin coated tubes and centrifuged (3850 × g, 15 min, 4 °C). Plasma cytokine levels were analyzed with a customized Bio-Plex Pro Assay (Bio‐Rad Laboratories; multiplexed: IL-1-β (171G5002M), TNF-α (171G5023M), IL-10 (171G5009M), IL-6 (171G5007M)) and Luminex 200 system (Merck laboratories).
8
Quantifying TNF-α in Osteoarthritic Chondrocytes
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Stored supernatants from cytokine-treated osteoarthritic chondrocytes incubated with various test substances were analyzed for the level of tumor necrosis factor-α [TNF-α] using the Bio-Plex Pro Assay and the Bio-Plex 200 analyzer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). In this cytokine multiplex assay, antibodies are covalently coupled to magnetic beads with a unique fluorescence dye. Thus, the concentrations of each analyte can be determined. The value below the lower limit of detection for the analyte was recorded as the lower limit of quantification (LLOQ). For example, analyzed TNF-α had an LLOQ of 3.33 pg/mL. In addition, the volume of every sample supernatant was measured to quantify proteins.
9
Antigen-Specific Cytokine Profiling
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To measure antigen-specific cytokine production, we aliquoted 100 μl of splenocyte suspension (1 × 106 cells/well) into each well of a 96-well plate and cultured them for 72 h using the same protocol as above, except BrdU was not used. The concentrations of four cytokines/chemokines, interleukin (IL)-2, IL-17, interferon (IFN)-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF), in the supernatants were measured using a Bio-plex Pro™ Assay (M60-009RDPD; Bio-Rad) according to the manufacturer’s instructions. The cytokine/chemokine levels were calculated by referring to a standard curve for each molecule derived using various concentrations of standards assayed in the same manner as the supernatant samples. The detection limit for each molecule was determined by recovering the corresponding standard, and the lowest values with > 70% recovery were set as the lower detection limits. No samples were beyond the upper detection limits, and some samples were below the lower detection limits.
10
Biomarker profiling in postpartum women
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Plasma and serum were collected at baseline and at the 6 months of follow‐up visit. Aliquots were stored at −80°C. N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) plasma levels were measured by routine laboratory workup as described.22 The measurement of plasma Gal3 (Human Galectin‐3 Platinum Elisa, eBiosciences, San Diego, USA), sST2 (Presage® ST2 Assay, Critical Diagnostics, San Diego, USA), serum PINP (PINP Elisa Assay, Cloud‐Clone Corp, Texas, USA), and serum PIIINP (PIIINP Elisa Assay, Cloud‐Clone Corp) was conducted according to the manufacturer's instructions. OPN was measured using Bio‐Plex Pro Assay (Human Cancer Biomarker Panel 1, Bio‐Rad, Hercules. USA).
In addition, cleavage of OPN by thrombin was measured by western blot [Anti‐Human Osteopontin (O‐17) Rabbit IgG Affinity Purify, Immuno‐Biological Laboratories Co. Japan].
We recruited 96 healthy pregnancy‐matched women without any history of cardiac disease and with a normal physical examination. Forty controls were recruited in SA, of whom 17 women were in the prepartum period and 23 were in the early postpartum period. Fifty‐six healthy women were recruited in Germany. All German controls were in the postpartum period.
In addition, cleavage of OPN by thrombin was measured by western blot [Anti‐Human Osteopontin (O‐17) Rabbit IgG Affinity Purify, Immuno‐Biological Laboratories Co. Japan].
We recruited 96 healthy pregnancy‐matched women without any history of cardiac disease and with a normal physical examination. Forty controls were recruited in SA, of whom 17 women were in the prepartum period and 23 were in the early postpartum period. Fifty‐six healthy women were recruited in Germany. All German controls were in the postpartum period.
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