The prepared samples for the original lentil variety (L8) and its γ-irradiated mutant lines were analyzed for their DPP-IV inhibitory activities using a DPP-IV inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI, USA). The kit provides a fluorescence-based method for screening DPP-IV inhibitors and the assay procedure is described in our previous studies [48 (link),49 (link)].
Dpp 4 inhibitor screening assay kit
Manufactured by Cayman Chemical
Sourced in United States, Estonia
The DPP-IV inhibitor screening assay kit is a laboratory tool designed to measure the inhibitory activity of compounds against the enzyme dipeptidyl peptidase-IV (DPP-IV). DPP-IV is an enzyme that plays a role in regulating various physiological processes. The kit provides a standardized method for screening and evaluating the potency of potential DPP-IV inhibitors.
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Lab products found in correlation
Clariostar, by BMG Labtech (2 mentions)
Sitagliptin, by Merck Group (2 mentions)
Synergy h1 fluorescent plate reader, by Agilent Technologies (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ace from porcine kidney, by Merck Group (1 mentions)
Acetonitrile acn, by Merck Group (1 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
Gly pro amido 4 methylcoumarin hydrobromide gly pro amc, by Merck Group (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Synergy h1 microplate reader, by Agilent Technologies (1 mentions)
4 nitrophenyl β d glucopyranoside, by Merck Group (1 mentions)
Hplc grade ethanol, by Merck Group (1 mentions)
Acarbose, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Prism software, by GraphPad (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
Prism 9, by GraphPad (1 mentions)
16 protocols using dpp 4 inhibitor screening assay kit
1
Lentil Mutant DPP-IV Inhibition Assay
2
Evaluating DPP-IV Inhibitory Activity
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The in vitro assessment of DPP-IV-inhibitory activity was performed following the manufacturer instructions (DPP-IV inhibitor screening assay kit–Cayman Chemical) and previously reported methods (47 (link), 48 (link)). The experiments were carried out in triplicate in a half–area 96 well solid plate (white). Each reaction (50 μL) was prepared adding the reagents in the following order in a microcentrifuge tube: 1 X assay buffer [20 mM Tris–HCl, pH 8.0, containing 100 mM NaCl, and 1 mM EDTA] (30 μL), CH and CH (F3) at final concentration range of 0.01–2.0 mg/mL (10 μL) or vehicle (10 μL of distilled H2O) and finally the DPP-IV enzyme (10 μL). Subsequently, the samples were mixed and 50 μL of each reaction were transferred in each well of the plate. The reactions were started by adding 50 μL of substrate solution to each well and incubated at 37°C for 30 min. Fluorescence signals were measured using the Synergy H1 fluorescent plate reader from Biotek (excitation and emission wavelengths 360 and 465 nm, respectively).
3
Oat Protein Isolates for DPP-IV Inhibition
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Protein isolates from all seven oat varieties and selected synthesised peptides were tested in vitro for DPP-IV inhibition. The bioassay (DPP-IV Inhibitor Screening Assay Kit, Cayman Chemical, Ann Arbor, MI, USA) was carried out as per the manufacturer’s instructions. First, 10 μL of each sample inhibitor at a concentration of 1 mg/mL assay buffer was added to 30 μL diluted assay buffer, 10 μL diluted DPP-IV, and 50 μL substrate solution, in triplicate. Samples were incubated at 37 °C for 30 min. Fluorescence was read with excitation wavelengths of 355 nm and emission wavelengths of 460 nm using a FLUOstar Omega microplate reader (BMG LABTECH GmbH, Offenburg, Germany). The percentage inhibition was calculated using the following equation:
4
DPP-IV Inhibitor Screening Assay
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DPP-IV inhibitor screening assay kit was obtained from Cayman Chemicals (Catalog no. 700210) and stored at −80 °C, DMSO (Sigma Aldrich).
5
Fluorometric Assay of DPP-IV Inhibitors
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A fluorometric assay kit (DPP (IV) Inhibitor Screening Assay Kit, Cayman chemical) was used to determine the DPP-IV inhibitory activity of compounds 6–10 . Sitagliptin (as part of a kit) was used as positive control inhibitor. Substances were diluted in dimethylsulfoxide (DMSO (Reagent Component, Moscow, Russia)) and tested at a final concentration of 100 µM (in 10% DMSO). For two compounds (7b , 9a ), inhibitory activity was also determined at concentrations of 0.1, 1, 10 and 1000 µM to estimate IC50. The assay was based on liberation of AMC (7-amino-4-methyl-coumarin) from DPP-IV substrate, Gly-Pro-AMC. Changes in fluorescence due to cleavage of the molecule by DPP-IV were measured with an excitation and emission wavelength at 350 and 450 nm using a ClarioStar (BMG Labtech, Germany). Percentage inhibition of DPP-IV activity was calculated relative to baseline activity (control group, without inhibitors) using the formula: %inhibition = (baseline activity − activity with inhibitor) / baseline activity * 100. IC50 (for compounds 7b and 9a ) was calculated using a four-parameter logistic regression model.
6
Casein Hydrolysate Bioactive Peptides
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Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin/streptomycin and 96-well plates were purchased from Euroclone (Milan, Italy). Acetonitrile (ACN), formic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sitagliptin, Gly-Pro-amido-4-methylcoumarin hydrobromide (Gly-Pro-AMC) and ACE from porcine kidney were from Sigma-Aldrich (St. Louis, MO, United States). ACE1 Activity Assay Kits come from Biovision (Milpitas Blvd., Milpitias, CA, United States). The DPP-IV inhibitor screening assay kit was bought from Cayman Chemical (Michigan, United States). Caco-2 cells were obtained from INSERM (Paris, France). Casein hydrolysates (CH), which is spray dried samples from the production process, was supplied by A. Costantino S.R.L. (Italy). (Casein hydrolysate sport/health batch 218F0004, 100 g). Casein hydrolysates (CH), which is spray dried samples from the production process, was supplied by A. Costantino S.R.L. (Italy). (Casein hydrolysate sport/health batch 218F0004, 100 g).
7
Fluorometric DPP-IV Inhibitor Screening Assay
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The DPP-IV assay was performed using a DPP-IV Inhibitor Screening Assay Kit (Cayman Chemical Item No. 700210, Tallinn, Estonia), according to the manufacturer’s instructions. This method uses the fluorogenic substrate Gly-Pro-Aminomethylcoumarin for measuring DPP-IV activity. We mixed 10 μL aliquots of the lipid extract with 10 μL of DPP-IV and 30 μL of assay buffer. The reaction started after the addition of 50 μL of substrate solution (100 mM of Gly-Pro-p-nitroanilide in Tris-HCl buffer (pH = 8.0)) to the microplate and incubated at 37 °C for 30 min; the resulting fluorescence was analyzed using an excitation wavelength of 350–360 nm and an emission wavelength of 450–465 nm (Synergy H1, Biotek Instruments, Winooski, VT, USA). Sitagliptin was the positive control inhibitor (10–100 μM). The analyses were performed in triplicate.
The background fluorescence was subtracted from the 100% initial activity for each inhibitor well. The inhibition rates (%) of the enzymes were calculated as follows:
The background fluorescence was subtracted from the 100% initial activity for each inhibitor well. The inhibition rates (%) of the enzymes were calculated as follows:
8
DPP4 Inhibitory Activity Assay
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DPP4 inhibitory activity was measured using a DPP(IV) inhibitor screening assay kit (Cayman Chemical Company) according to the manufacturer's protocol, with minor modifications. Briefly, the enzyme solution (120 µl) was dissolved in 480 µl DPP assay buffer [20 mM Tris-HCI (pH 8.0), 100 mM NaCl and 1 mM EDTA] and was used as the enzyme solution. The substrate, 5 mM H-Gly-Pro conjugated with aminomethylcoumarin (AMC), was prepared in the same buffer. The assay procedure was performed according to the manufacturer's protocols, and is briefly described as follows: Diluted assay buffer (30 µl) and diluted enzyme solution (10 µl) were added to the 96-well plates containing 10 µl solvent (blank) or solvent-dissolved test compounds (0-75 µM). The reaction was initiated by adding 50 µl diluted substrate solution, the reaction was measured using fluorometric determination (excitation wavelength, 350 nm; emission wavelength, 450 nm) using a plate reader (Tecan Group, Ltd.) every 30 sec for 20 min at 37˚C. Sitagliptin, which was included in the kit, was used as a positive control. Various concentrations (0-75 µM) of the enzyme inhibitor (tested compounds) and substrate were used in the reactions, the initial rate of reaction (v) based on free AMC, and the release rate were calculated using Lineweaver-Burk [1/v vs. 1/(substrate)] or Dixon plots [1/v vs. (inhibitor)].
9
Screening Assay for DPP-IV Inhibitors
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The assay was performed with a commercial DPP (IV) inhibitor screening assay kit (Cat. 700210, Cayman Chemical, Tallinn, Estonia). Briefly, 10 μL aliquots of different concentrations of the active extracts were mixed with 10 μL of DPP-IV and 30 μL of assay buffer. After the addition of 50 μL of substrate solution (100 mM of Gly-Pro-p-nitroanilide in Tris-HCl buffer (pH = 8.0)) to initiate the reaction, the plate was incubated for 30 min at 37 °C, and the fluorescence was read using an excitation wavelength of 350–360 nm and an emission wavelength of 450–465 nm (Synergy H1 microplate reader, Biotek, Winooski, VT, USA). Sitagliptin was used as the positive control (10–100 μM).
The enzyme and sample solution of the sample control group and the enzyme solution of the blank control group were replaced with buffer, whereas all the remaining reagents were kept equal to those of the sample group.
The background fluorescence was subtracted to the 100% initial activity for each inhibitor well. The inhibition rates of the enzymes were calculated according to Equation (4).
The enzyme and sample solution of the sample control group and the enzyme solution of the blank control group were replaced with buffer, whereas all the remaining reagents were kept equal to those of the sample group.
The background fluorescence was subtracted to the 100% initial activity for each inhibitor well. The inhibition rates of the enzymes were calculated according to Equation (4).
10
Fluorometric Assay for DPP4 Inhibition
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A fluorometric screening assay kit (DPP (IV) Inhibitor Screening Assay Kit, Cayman Chemical, Ann Arbor, MI, USA) was used to determine the inhibitory activity of QS-528 and QS-619 against DPP4. Sitagliptin (as part of the kit) was used as a positive control. The compounds were diluted in dimethylsulphoxide (DMSO (Reagent Component, Moscow, Russia)) and tested at a final concentration of 100 µM (in 10% DMSO). The assay was based on the liberation of AMC (7-amino-4-methyl-coumarin) from the DPP4 substrate, Gly-Pro-AMC. Fluorescence changes resulting from the cleavage of the DPP4 molecule were measured at excitation and emission wavelengths of 350 and 450 nm using ClarioStar (BMG Labtech, Ortenberg, Germany). The percentage inhibition of DPP4 activity was calculated relative to baseline activity (control group, without inhibitors) using the formula that follows: %inhibition = (baseline activity − activity with inhibitor)/baseline activity × 100.
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