Serum RBP4 was determined by the enzyme-linked immunosorbent assay (ELISA) method using Human RBP4 Quantikine ELISA Kits (No. Cat. DRB400, R&D Systems, Inc., Minneapolis, Minnesota, United States). The experimental steps are briefly summarized as follows: first, standards or samples were added to the precoated microplates and incubated for 1 h at 37°C. Next, the antibody conjugate was added and incubated for 1 h at 37°C. After washing, substrate solution was added and further incubated for 30 min at 37°C. Finally, stop solution was added, and the intensity of the color was read at 450 nm within 30 min using a Multiskan spectrometer (Thermo Fisher Scientific, Inc., Waltham, Massachusetts, United States). Samples were tested in triplicate.
Multiskan spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Multiskan spectrometer is a microplate-based absorbance reader that measures the optical density of samples in microplates. It is designed to perform a variety of absorbance-based assays, including enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and other colorimetric measurements.
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Lab products found in correlation
Combiflash rf 200i, by Teledyne (1 mentions)
Avance 500 mhz, by Bruker (1 mentions)
6 nbdg, by Thermo Fisher Scientific (1 mentions)
Agilent 6520 accurate mass q tof lc ms, by Agilent Technologies (1 mentions)
Streptozotocin (stz), by Sisco Research Laboratories (1 mentions)
Metformin, by Merck Group (1 mentions)
α glucosidase, by Sisco Research Laboratories (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Lc 20ad, by Shimadzu (1 mentions)
4 protocols using multiskan spectrometer
1
Quantification of Serum RBP4 by ELISA
2
Nanoparticle Drug Loading and Encapsulation Efficiency
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The drug loading content (LC) and drug encapsulation efficiency (EE) of the SFB-loaded NPs were determined as described (Lin et al., 2016 (link)). SFB-loaded NPs were centrifuged at 14,000 rpm for 30 min, and the supernatant fractions were collected and analyzed by UV spectroscopy (Multiskan Spectrometer, Thermo Scientific, Rockford, IL) at 270 nm. The vehicle background absorption was subtracted by measuring the absorption of the supernatants of NPs prepared without SFB under the same conditions. All measurements were performed in triplicate. The LC and EE values of the SFB-loaded NPs were calculated using the following formulas:
3
Hemolytic Activity Assay for Peptides
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A haemolytic activity assay was used for determining whether peptides acted on hRBC membrane by calculating the minimum haemolytic concentration (MHC). Briefly, 100 µL serial dilutions of peptides were incubated in a 96-well plate with an equal volume of a fresh hRBC suspension at 5% v/v in 1X PBS (200–0.39 µM final peptide concentrations). The samples were spun at 1000× g for 5 min after having been incubated at 37 °C for 1 h and a Multiskan spectrometer (Thermo Fisher) was used for measuring 100 µL supernatant absorbance at 560 nm. Treated hRBC absorbance 0.1% Triton X-100 or 100% haemolysis was used as positive control [46 (link)].
4
Purification and Characterization of Bioactive Compounds
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Combiflash Rf 200i (Teledyne ISCO) fitted with glass column and silica gel 60 RP-18 (40–63 μm) were used for purification of fractions. Shimadzu HPLC, LC 20AD with PDA detector fitted with either an analytical column (Discovery RP Amide C-16 Supelco 5 μm 250 × 4.6 mm) or semi-preparative (Discovery Amide C-16 Supelco 5 μm 250 × 10 mm) were used for analysis and purification of compounds. Column chromatography was carried out with Merck silica gel (100–200 mesh size). Thin layer chromatography (TLC) was done on TLC plates pre-coated with silica gel 60F254 (0.25 mm normal phase Merck). α-Glucosidase (Maltase EC 3.2.1.20) and p-nitro phenyl-α-d -glucopyranoside were purchased from Sisco Research Laboratory (SRL). Streptozotocin was purchased from Sisco Research Laboratories, Mumbai and metformin from Sigma Aldrich USA. 6-NBDG was procured from Invitrogen. NMR spectra were recorded on a Bruker Avance 500 MHz instrument with TMS as internal standard. Chemical shifts are expressed in δ values. Agilent 6520 Accurate mass Q-TOF/LC-MS was used to determine molecular weight. Absorbance was measured by Thermo Scientific Multiskan spectrometer. All other chemicals and reagents were of analytical grade.
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