Plasma adiponectin concentrations were measured using an enzyme immunoassay (R&D Systems, Minneapolis, USA, DRP300). The assays were performed in duplicate using a microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA) set to 450 nm (intra-assay and inter-assay variations were less than 8% and 5%, respectively). Serum levels of mature-BDNF were measured using an ELISA Kit (Aviscera bioscience, Santa Clara, CA, USA, SK00752-01). The assay was performed in duplicate using a microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA) set to 450 nm (intra-assay and inter-assay variations were less than 10% and 6%, respectively).
Microplate absorbance reader
Manufactured by Bio-Rad
Sourced in United States, Japan
The Microplate absorbance reader is a laboratory instrument designed to measure the optical density or absorbance of samples in a microplate format. It can be used to quantify various analytes in a wide range of applications, including enzyme-linked immunosorbent assays (ELISA), cell-based assays, and colorimetric assays.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting kit 8 cck 8, by Dojindo Laboratories (6 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (6 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (4 mentions)
Cck 8, by Dojindo Laboratories (4 mentions)
Cck 8 reagent, by Dojindo Laboratories (4 mentions)
Cck 8 reagent, by Beyotime (4 mentions)
Mcx130, by R&D Systems (3 mentions)
Mje00b, by R&D Systems (3 mentions)
Mtt cell viability assay kit, by Thermo Fisher Scientific (3 mentions)
Dtm100, by R&D Systems (2 mentions)
Cytotoxicity detection kit, by Roche (2 mentions)
Cck 8 solution, by Dojindo Laboratories (2 mentions)
Cell counting kit 8 cck 8, by Beyotime (2 mentions)
Matrigel, by BD (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Beyotime (2 mentions)
Cck 8 assay, by Dojindo Laboratories (2 mentions)
Drp300, by R&D Systems (1 mentions)
Dcp00, by R&D Systems (1 mentions)
Dtm200, by R&D Systems (1 mentions)
48 well culture plates, by BD (1 mentions)
128 protocols using microplate absorbance reader
1
Measuring Adiponectin and BDNF Levels
2
Quantifying Cell Secreted Proteins
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Cell-secreted TIMP-1 (DTM100, R&D Systems, Minneapolis, MN, USA), TIMP-2 (DTM200, R&D Systems, Minneapolis, MN, USA), MCP-1 (DCP00, R&D Systems, Minneapolis, MN, USA), and OPG (RAB0484, Sigma-Aldrich, St. Louis, Missouri, USA) were measured with the respective ELISA kits containing pre-coated ELISA plates, and the assays were performed as described by the manufacturers. Briefly, 100 μL of cell culture supernatant or standard was incubated in each well for 3 h at RT. Then, the HRP-conjugated antibody was added and incubated for 1 h at RT, followed by aspiration and three washes. Next, horseradish peroxidase was added and incubated for 1 h, followed by aspiration and washes. 3,3′,5,5′-Tetramethylbenzidine substrate was added to each well and incubated for 30 min in a dark chamber. Stop solution was added, and the absorbency of all ELISAs was read at 450 nm with a plate reader (Bio-Rad® Microplate Absorbance Reader, Bio-Rad Laboratories Inc., Hercules, CA, USA).
3
ELISA Verification of Biomarker Candidates
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ELISA was conducted to verify the biomarker candidate proteins identified in transcriptomic and proteomic analyses. An additional 12 AH samples from each group were subjected to ELISA. AH proteins were measured using the human metallopeptidase inhibitor 1 (TIMP1) (DTM100; R&D Systems), angiopoietin-related protein 7 (ANGPTL7), and Fc fragment of IgG binding protein (FCGBP) ELISA Kit (CSB-EL001715HU, CSB-EL008536HU; Cusabio Technology) according to the manufacturer’s instructions [48 (link),49 (link)]. Briefly, all samples were brought to room temperature before use and were assayed in duplicate. 50 μL of samples or standard was added into the wells pre-coated with an antibody specific to the antigen (human TIMP-1, human ANGPTL7, or human FCGBP) and incubated for 2 h at room temperature. This was followed by incubation for 2 h with the target antibody conjugates. Substrate solution was added to the samples and was incubated for 30 min. Stop solution was added, and the absorbance of color at 450 nm was measured using a microplate reader (Bio-Rad® Microplate Absorbance Reader, Bio-Rad Laboratories Inc., Hercules, CA, USA). The intra-assay and inter-assay coefficients of variation within and between ELISA tests were <8%. All absorbance results are expressed as nanogram per milliliter.
4
Azocasein Protease Activity Assay
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Fifty μL of a 1% (w/v) azocasein solution were added to 50 μL of cell-free supernatant (containing protease) and the mixture was immediately incubated at 37 °C for 30 min. The reaction was stopped by mixing with 300 μL of 5% (w/v) trichloroacetic acid (TCA, Katayama Chemical, Osaka, Japan). Finally, the sample was centrifuged at 13,000 rpm for 10 min and 150 µL of the supernatant was mixed with an equal amount of 0.5 N NaOH. The absorbance of the mixture was measured at 450 nm using a Microplate Absorbance Reader (BIO-RAD, Hercules, CA, USA). One unit was defined as an increase of A450 nm of 0.01 after incubation for 1 min [52 (link),53 (link),54 (link)].
5
Cell Proliferation Assay with CCK-8
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To measure the proliferation capacity, the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) and the Passage 4 (P4) cells were used as described previously [27 (link)]. Briefly, 10 μL of CCK-8 solution was added to each well of a 96-well plate containing 5000 cells. After incubating the plate at 37 °C for 4 h, absorbance at 450 nm was measured using a microplate absorbance reader (BioRad, USA). Cell proliferation was tested on days 1, 3, 5, 7, 9, 11 and 13. A blank 96-well plate was used for the zero setting. All experiments were performed thrice.
6
Cell Viability Assay Protocol
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The cell viability was examined using a Cell Counting Kit-8 (CCK8, R&S Biotechnology, Shanghai, China) reagent. In short, TPC and BCPAP cells were plated into 96-well plates (1×103/well). After 0, 24, 48, and 72 h, each well was mixed with a CCK-8 solution (10 μL) and incubated at 37 °C for an additional 2 h. A microplate absorbance reader (Bio-Rad Laboratories, CA) was used to measure the absorbance (wavelength: 450 nm).
7
Soluble Aβ Fractionation and Chemokine Quantification
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Hippocampal lysates from Gas6 and control-treated APP/PS1 mice were centrifuged at 100,000 g for 1 h at 4 °C to separate soluble aggregations of Aβ (monomers and oligomers) from large, insoluble Aβ fibrils. Supernatants were collected as the soluble fragments. CXCL13 and CCL2 chemokine levels were measured utilizing respective mouse ELISA kits (R&D MCX130 and MJE00B). Soluble samples were diluted 1:1 in kit buffer. Plates were read with Microplate Absorbance Reader (Bio-Rad). Linear regression models were used for CXCL13 & CCL2.
8
Soluble Aβ Fractionation and Chemokine Quantification
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Hippocampal lysates from Gas6 and control-treated APP/PS1 mice were centrifuged at 100,000 g for 1 h at 4 °C to separate soluble aggregations of Aβ (monomers and oligomers) from large, insoluble Aβ fibrils. Supernatants were collected as the soluble fragments. CXCL13 and CCL2 chemokine levels were measured utilizing respective mouse ELISA kits (R&D MCX130 and MJE00B). Soluble samples were diluted 1:1 in kit buffer. Plates were read with Microplate Absorbance Reader (Bio-Rad). Linear regression models were used for CXCL13 & CCL2.
9
Quantitative GLP-1 ELISA Protocol
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The plasma GLP-1 level was measured using a commercially available quantitative Total ELISA kit (EMD Millipore, Billerica, MA, USA), with the microplate absorbance reader (Bio-Rad) set to 450 nm. The assay was performed in duplicate.
10
Cell Proliferation Assay with CCK-8
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Cell proliferation was detected with the Cell Counting Kit-8 (Dojindo Laboratories, Japan) assay according to the manufacturer's protocol. Initially, 3 × 104 RA-FLSs per well were seeded into 96-well plates and incubated to 70%–90% confluence. Cells in logarithmic growth phase were then transfected according to the above method. Three triplicate wells were applied to cell culture for another 24, 48, 72, and 96 h in each group. At each experimental point, 10 μL CCK-8 solution was added to each well and cells were incubated at 37 °C for 1.5 h. Cell viability was evaluated by absorbance at 450 nm measured with a microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA).
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