Synergy h1 fluorescent plate reader

Total RNA was used for the cDNA library construction using TruSeq® Stranded mRNA LT kit (Illumina, San Diego, USA) and epMotion 5075t robot (Eppendorf, Hamburg, Germany). Library construction produced single-indexed libraries with a median insert size of ∼300 bp which was validated on an Agilent 2200 TapeStation instrument using D1000 ScreenTapes (Santa Clara, CA, USA). All libraries were quantified in triplicate using SynergyH1 fluorescent plate reader (BioTek, Vermont, USA). The pooled denatured cDNA libraries were loaded on a cBot for cluster generation followed by 2 × 108 bp paired-end sequencing using HiSeq Rapid kits with V2 chemistry on an HiSeq 2500 sequencer (Illumina, San Diego, USA). All the sequence files were uploaded into the NCBI-Sequence Read Archive (SRA) and Bio project ID: PRJNA517543 and used for data analysis.

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).

The in vitro experiments were carried out in triplicate in a half volume 96 well solid plate (white). Each reaction (100 μL) was prepared by adding the reagents in a micro centrifuge tube in the following order: 1 × assay buffer [20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl, and 1 mM EDTA]; PBP hydrolysate with a final concentration of 0.1, 0.5, 1.0, 2.5, or 5.0 mg/mL, or vehicle; and finally, the purified human DPP-IV enzyme (10 μL, from Cayman Chemical Company, Ann Arbor, MI, USA). Subsequently, the samples were mixed, and 50 μL of each reaction were transferred into each plate well. Each reaction was started by adding 50 μL of substrate solution (200 µM H-Gly-Pro-7-amido-4-methylcoumarin (AMC)) to each well and incubated at 37 °C for 30 min. Fluorescence signals were measured using the Synergy H1 fluorescent plate reader (Biotek, Bad Friedrichshall, Germany) (excitation and emission wavelengths 360 and 465 nm, respectively).

HepG2 cells (3.0 × 10⁴/well) were seeded in black 96-well plates and kept in complete growth medium for 2 d before treatment. The third day, they were treated with 4.0 μg/mL PCSK9 and 4.0 μg/mL PCSK9 + peptides (0.5-50.0 µM), and vehicle (H₂O) for 2 h with at 37 °C under 5% CO₂ atmosphere. At the end of the treatments, the culture medium was replaced with 50.0 μl/well LDL-DyLight™ 550 working solution (Cayman Chemical Company, Ann Arbor, MI, US). The cells were additionally incubated for 2 h at 37 °C and then the culture medium was aspirated and replaced with PBS (100.0 μl/well). The degree of LDL uptake was measured using the Synergy H1 fluorescent plate reader from Biotek (excitation and emission wavelengths 540 and 570 nm, respectively).

HepG2 cells (3 × 10⁴/well) were seeded in 96-well plates and kept in complete growth medium for 2 d before treatment. The third day, they were treated with T9 (160 and 320 μM) or P5 (10 μM), or vehicle (H₂O) for 24 h. At the end of the treatment, the culture medium was replaced with 50 μl/well LDL-DyLight™ 550 working solution (Cayman Chemical Company, Ann Arbor, MI, US). The cells were additionally incubated for 2 h at 37 °C and then the culture medium was aspirated and replaced with PBS (100 μl/well). The degree of LDL uptake was measured using the Synergy H1 fluorescent plate reader from Biotek (excitation and emission wavelengths 540 and 570 nm, respectively).

Four independent in
vitro experiments were carried out, and each of those was performed
in triplicate in a white half-volume 96-well solid plate. Each reaction
(50 μL) was prepared by adding the reagents in a microcentrifuge
tube in the following order: 1× assay buffer [20 mM tris–HCl,
pH 8.0, containing 100 mM NaCl and 1 mM EDTA] (30 μL), final
compounds (1–14) (from 10^–10 to 10^–3 M) or vehicle (10 μL), and finally
the DPP IV enzyme (10 μL). Afterward, the samples were mixed
and 50 μL was transferred to each plate well. Each reaction
was started by adding 50 μL of the substrate solution (200 μM
H-Gly-Pro-7-amido-4-methylcoumarin (AMC)) to each well and incubated
at 37 °C for 30 min. Fluorescence signals were measured using
a Synergy H1 fluorescent plate reader from Biotek (excitation and
emission wavelengths 360 and 465 nm, respectively). The DPP IV enzyme
and the substrate solution were provided by Cayman Chemicals (Michigan).

Caco-2 cells (5 × 10⁴/well) were seeded in black 96-well plates with clear bottoms and cultured for 24 h. Afterwards, spent media was removed and CP and CT hydrolysates (1.0, 2.5, and 5.0 mg/mL), sitagliptin at 1.0 μM (positive control), or vehicle in growth medium were separately used to treat Caco-2 cells for 24 h at 37 °C. Treatment media were then removed and cells were washed with 100 μL of PBS without Ca⁺⁺ and Mg⁺⁺, and 100 μL of Gly-Pro-AMC substrate (Cayman Chemical, Ann Arbor, MI, USA) at the concentration of 50.0 μM in PBS without Ca⁺⁺ and Mg⁺⁺ were added in each well. Fluorescence signals (ex./em. 350/450 nm) were detected using the Synergy H1 fluorescent plate reader from Biotek every 1 min for 10 min.