To investigate the effects of RBE on Akt1 signaling in adipocytes, 3T3-L1 cells were cultured and differentiated in 6-well plate. After treatment with RBE for 8 days, cells were washed with cold PBS and lysed with 200 µL/well of ice-cold 1X MILLIPLEX® MAP lysis buffer (EMD Millipore, Merck, Germany). Cell lysates were gently rocked for 15 min at 4 °C and centrifuged at 14,000× g under 4 °C for 15 min. Supernatants were collected and stored at −80 °C for further experiments. The phosphorylation levels of Akt1 (Ser473) were determined using the MILLIPLEX® MAP Phospho/Total Akt1 2-plex Magnetic Bead Panel kit (EMD Millipore, Merck, Germany) according to the manufacturer’s instruction. The fluorescence intensity of the beads was measured and analyzed using the Luminex® system (EMD Millipore, Merck, Germany). Data were normalized to the total protein concentration from the BCA kit (Thermo Fisher, USA) using BSA as a standard. The results were expressed as the Median Fluorescence Intensity (MFI) per mg protein (MFI/mg protein).
Luminex system
Manufactured by Merck Group
Sourced in United States, Germany
The Luminex system is a multiplex detection platform that utilizes flow cytometry technology to simultaneously analyze multiple analytes in a single sample. The system is designed to perform various assays, including protein, nucleic acid, and cell-based analyses. The Luminex system employs color-coded microspheres, also known as beads, to which specific capture reagents are coupled, enabling the detection and quantification of multiple targets in a single reaction.
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Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Mice adipokine multiplex kit, by Merck Group (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Milliplex map 9 plex multi pathway magnetic bead signaling kit, by Merck Group (1 mentions)
Milliplex map, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Cell lysis buffer, by Merck Group (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Milliplex map technology, by Merck Group (1 mentions)
Primepcr technology, by Bio-Rad (1 mentions)
Microtainer serum separator tube, by BD (1 mentions)
Cfx connect thermocycler, by Bio-Rad (1 mentions)
Iscript, by Bio-Rad (1 mentions)
12 protocols using luminex system
1
Akt1 Signaling in Adipocytes under RBE
2
Cytokine Profiling in Liver Cells
3
Cellular Protein Analysis Protocol
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The studies were carried out according to the standard protocol. M-HeLa cells were incubated for 24 h with the test substance. Cells were lysed in MILLIPLEX® MAP Lysis buffer containing protease inhibitors. A total of 20 μg of total protein of each lysate diluted in MILLIPLEX® MAP Assay Buffer 2 was analyzed according to the analysis protocol (the lysate was incubated at 4 °C overnight). The mean fluorescence intensity (MFI) was detected using Luminex® system, MERCK, Kenilworth, NJ, USA.
4
Phosphorylation Dynamics of Key Signaling Pathways
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HAECs and HASMCs were grown until confluent and treated with ucOCN (10 ng/ml) or with control media for 2, 5, 10, and 30 min. The MILLIPLEX MAP 9‐plex Multi‐Pathway Magnetic Bead Signaling Kit (48‐680MAG; Merck Millipore) was performed according to the manufacturers’ instructions to detect changes in phosphorylated ERK/MAP kinase 1/2 (Thr185/Tyr187), AKT (Ser473), STAT3 (Ser727), JNK (Thr183/Tyr185), p70s6 kinase (Thr412), NF‐kB (Ser536), STAT5A/B (Tyr694/699), CREB (Ser133), and p38 (Thr180/Tyr182) in cell lysates using Luminex® xMAP® technology. Phosphorylated‐ and total‐mTOR (Ser2448) were also measured in the lysate samples using the Luminex system (48‐625MAG, Merck Millipore).
5
Hormone levels in macroH2A1.2 Tg mice
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Insulin, leptin and IGF-1 levels were assessed in the sera of wild-type and macroH2A1.2 Tg animals, using a customized mouse MILLIPLEX® MAP (multi-analyte panels) Luminex system (Merck Millipore) [22 (link)], according to the manufacturer’s instructions.
6
Insulin Resistance Measurement in Mice
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Mice were fasted overnight and plasma samples were obtained to analyze insulin (Mice Adipokine Multiplex Kit, Millipore, St. Charles, MO, USA) concentrations using the Bio-Rad Luminex system (Hercules, CA, USA) and Millipore kits (St. Charles, MO, USA) according to manufacturer’s protocols. The homeostatic model assessment of insulin resistance was calculated as glucose concentration (mmol/L)×insulin concentration (mU/L)/22.5 [32 (link)].
7
Plasma Cytokine Quantification Protocol
8
Cytokine Quantification in Biological Samples
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Freshly obtained samples were weighed and homogenized in ice-cold PBS supplemented with protease inhibitor cocktail (Roche). The protein concentration was measured by BCA kits. The concentrations of cytokines were determined by Milliplex Multiplex Assays Using Luminex system (Millipore) or commercially available ELISA kits (R&D Systems) in accordance with the manufacturer’s protocols. The detection limits of individual cytokines are as follows: IL-8 (0.34–3425 pg/mL), IFN-β (0.84–13,254 pg/mL), IL-1β (1.09–23,091 pg/mL), IFN-γ (2.79–56,496 pg/mL), TRAIL (1.86–24,073 pg/mL), IL-1α (0.49–4349 pg/mL), FasL (0.57–9219 pg/mL), CXCL1 (23.01–22,008 pg/mL), TNF-α (0.50–10,285 pg/mL), IL-6 (0.30–6113 pg/mL), HMGB1 (31.25–2000 pg/mL).
9
Cytokine Profiling in Mouse Eyes
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The mice were anesthetized, and their eyes were enucleated and placed in cell lysis buffer (Millipore Corporation, St. Charles, MO) containing a protease inhibitor mixture (Sigma-Aldrich Corp) for 20 min. After centrifugation at 15,000 × g for 15 min at 4 °C, the concentration of the supernatant was determined using a bicinchoninic acid protein assay kit (Beyotime, China). The concentrations of cytokines, including IL-1β, TNF-α, IL-4 and IL-10, were measured in duplicate using a Luminex system (Millipore Corporation., Germany) according to the manufacturer’s protocol. The results were normalized based on the protein concentration of the samples. In this part, 45 mice were used.
10
Insulin Resistance Measurement in Mice
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