Cerebral Aβ peptides and cytokines were quantified with the MSD V-PLEX Human Aβ42 Kit, the V-PLEX Plus Aβ Peptide Panel 1 (6E10) Kit and the V-PLEX Plus Proinflammatory Panel 1 Mouse Kit according to the manufacturer’s instructions (Meso Scale Discovery). Signals were measured with a SECTOR Imager 2400 reader (Meso Scale Discovery). Cerebral chemokines were quantified with Chemokine 9-Plex Mouse ProcartaPlex™ Panel 1, according to the manufacturer’s instructions (eBioscience). Data acquisition was performed with a MAGPIX (Luminex). Each sample was measured in duplicate. Aβ peptides, secreted cytokines and chemokines were normalized to the total brain protein concentration, evaluated with a BCA assay.
Sector imager 2400 reader
Manufactured by Mesoscale
Sourced in United States
The SECTOR Imager 2400 is a multipurpose reader designed for analyzing and quantifying various types of assays. It features a flexible optical system that can accommodate a range of microplate formats and detection modes, including chemiluminescence, fluorescence, and absorbance. The instrument is capable of rapid data acquisition and precise measurements, making it suitable for a wide variety of applications in research and diagnostic settings.
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Lab products found in correlation
V plex plus proinflammatory panel 1 mouse kit, by Mesoscale (2 mentions)
V plex plus aβ peptide panel 1 6e10 kit, by Mesoscale (1 mentions)
Anti her2, by Agilent Technologies (1 mentions)
Easysep mouse pan b cell isolation kit, by STEMCELL (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Nicotine, by Merck Group (1 mentions)
Cytometric bead array kit, by BD (1 mentions)
Facsverse analyzer, by BD (1 mentions)
Facsuite software, by BD (1 mentions)
Konelab 20 autoanalyzer, by Thermo Fisher Scientific (1 mentions)
Nuclear extraction kit, by Merck Group (1 mentions)
Precellys 24 homogeniser, by Avantor (1 mentions)
Protease phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
10 protocols using sector imager 2400 reader
1
Quantification of Cerebral Aβ, Cytokines, and Chemokines
2
Quantitative Amyloid-β Determination
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Quantitative determination of amyloid‐β was performed using an electrochemiluminescence ELISA for Aβ38, Aβ40, and Aβ42 according to the protocol of the supplier (Meso Scale Discovery, Rockville, MD, USA). Signals were measured on a SECTOR Imager 2400 reader (Meso Scale Discovery, Rockville, MD, USA).
3
Quantitative Analysis of HER-2 Expression
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In order to quantitate HER-2 expression values, we performed well-based reverse phase protein array (RPPA) as previously reported [13 (link)]. Briefly, extracted proteins (400 ng) were applied onto 96-well Multi-Spot™ plates (Meso Scale Discovery, Gaithersburg, MD, MA2400 96 HB Plate), the plate was allowed to dry at RT, and if needed, further incubated at 37°C for 30 min. The antigen-coated plates were pre-incubated with 5% BSA in PBST for 60 min at RT before primary antibody incubation. Anti-HER-2 (DAKO) and anti-GAPDH (Calbiochem) were diluted 1:1000 and 1:5000 with 5% BSA in PBST, followed overnight incubation at 4°C. After washing with PBST, the plates were incubated for 90 min with goat anti-rabbit or mouse SULFO-TAG™ antibodies (Meso Scale Discovery) at a dilution of 1:2000. The plates were washed three times with PBST. MSD-T read buffer was added to the plates and signals were detected using Sector Imager 2400 reader (Meso Scale Discovery). BSA coated wells were included on each plate as a control of non-specific binding. HER-2 expression signal was normalized with the value of GAPDH.
4
Measuring TNF mRNA and NF-κB Translocation in B Cells
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For measurements of TNF mRNA, FO B cells were sorted (the gating strategy is shown in fig. S4A) and stimulated with 2.5 μM CpG (InvivoGen) and nicotine (Sigma-Aldrich) in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin and 10 mM Hepes. After 4 hours of incubation, cells were collected, and pellets were solubilized in TRIzol reagent. These solution were stored at −80°C. For measurement of secreted TNFα, the culture supernatant was collected after 24 hours of incubation. TNFα concentration was measured with the V-PLEX Mouse TNF-α Kit (Meso Scale Diagnostics) and the Sector Imager 2400 Reader (Meso Scale Diagnostics) according to the manufacturer’s instructions.
For NF-κB translocation assays, B cells were isolated from naïve mouse spleens with the EasySep Mouse Pan-B cell Isolation Kit (STEMCELL Technologies) and suspended at 1.0 × 106 cells/ml in the supplemented RPMI 1640 (Gibco). Cells were stimulated with 1.0 μM CpG with or without nicotine for an hour. After washing, cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer.
For NF-κB translocation assays, B cells were isolated from naïve mouse spleens with the EasySep Mouse Pan-B cell Isolation Kit (STEMCELL Technologies) and suspended at 1.0 × 106 cells/ml in the supplemented RPMI 1640 (Gibco). Cells were stimulated with 1.0 μM CpG with or without nicotine for an hour. After washing, cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer.
5
Quantitative Assessment of Cerebral Cytokines
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Quantitative determination of cerebral cytokines was performed using an electrochemiluminescence ELISA for IFN‐γ; IL‐1β; IL‐2; IL‐4; IL‐5; IL‐6; CXCL‐1; IL‐10; IL‐12p70; TNF‐α mouse pro‐inflammatory panel kit according to the manufacturer's guidelines (Meso Scale Discovery, Rockville, MD, USA). Signals were measured on a SECTOR Imager 2400 reader (Meso Scale Discovery). Quantitative determination of cerebral CCL‐2, CCL‐3, and CCL‐4 chemokines was performed using BD Cytometric Bead Array Kit according to the manufacturer's guidelines (BD Biosciences). Data acquisition was performed using a FACSVerse analyzer (BD Biosciences). Fluorescence intensity of beads coated with chemokines coupled to phycoerythrin (PE) and samples was analyzed with BD FACSuite Software (BD Biosciences). Each sample was measured in duplicate.
Aβ plaque staining and immunohistochemistry were performed as detailed in Data S1.
Aβ plaque staining and immunohistochemistry were performed as detailed in Data S1.
6
Blood Lipid and Cytokine Analysis
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Blood was obtained after a 4 h fast the day before the mice were sacrificed. Cholesterol and triglycerides were measured on a Konelab 20 autoanalyzer (Thermo, Vantaa, Finland). Plasma cytokines were analyzed with a SECTOR Imager 2400 reader (Meso Scale Discovery, Gaithersburg, MD).
7
Cytokine Quantification via Sector Imager
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Cytokine levels in the media were analyzed with a SECTOR Imager 2400 reader (Meso Scale Discovery).
8
Cytokine and NF-kB Profiling in Mouse Brain
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Mice were sacrificed, and the corpus callosum was dissected and stored in liquid nitrogen. Samples were homogenised in PBS containing protease/phosphatase inhibitor (Thermo Scientific) using a Precellys 24 homogeniser (Peqlab), and lysed in RIPA buffer (25 mM TRIS, 150 mM NaCl, 1 % NP-40, 0.5 % Na-deoxycholate, 0.1 % SDS, pH 7.2). Quantitative cytokine determination was performed using an electrochemiluminescence ELISA (Mouse ProInflammatory Ultra-Sensitive Kit, Meso Scale Discovery). Signals were measured on a SECTOR Imager 2400 reader (Meso Scale Discovery). For NF-kB measurements, nuclear extracts were created from whole brain homogenates (Nuclear Extraction Kit, #2900, Millipore), and NF-kB activity in nuclear extracts was determined with a commercial assay (EZ-TFA assay, #70-610, Millipore) according to the manufacturer’s instructions using a microplate reader (FLUOstar Omega, BMG Labtech). All measurements were performed in duplicates.
9
Cytokine Profiling in Murine Fracture
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C57BL/6 mice were anaesthetized, and closed fractures were created in the lower limbs. Mice are culled at 3 h post-operatively, and the fractured bone segments are harvested and incubated in serum-free DMEM + 1% penicillin/streptomycin for 16 h. Cytokine levels were measured using a pro-inflammatory 7-plex plate and a SECTOR Imager 2400 reader (Mesoscale Discovery).
10
Pro-Inflammatory Cytokine Profiling in Mouse Brain
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Pro‐inflammatory response was determined in brain lysates using the V‐PLEX Plus Pro‐inflammatory Panel 1 (mouse) Kit for 10 cytokines (IFN‐γ, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, KC/GRO, IL‐10, IL‐12p70, and TNF‐α) following the protocol provided by the supplier (Meso Scale Discovery, Rockville, MD, USA). Briefly, 50 μl of diluted sample, calibrator, or control was added per well. The plate was sealed with an adhesive plate seal and incubated at room temperature with shaking for 2 h. Later, the plate was washed three times and the detection antibody was added. The plate was sealed and incubated at room temperature with shaking for 2 h. Finally, the plate was washed and the read buffer was added. Signals were measured on a SECTOR Imager 2400 reader (Meso Scale Discovery, Rockville, MD, USA).
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