Multilabel plate reader

A luciferase based 2 deoxyglucose assay was performed using human plaque VSMCs (Promega J1341). Briefly, cells were stimulated and quiesced in a zero glucose media. 2-deoxy glucose (1 mM) was pulse incubated with the cultures using a 96-well plate format for ∼10 min. Cells were then lysed and neutralized before a custom lucigenic reaction mixture was added. Luminescence was then recorded using a 0.3 integration interval on a VICTOR, Perkin Elmer multi-label plate reader.

The full-length recombinant protein of PDLIM1 was purchased from Cloud-Clone Corp. (Wuhan, China). PDLIM1 recombinant protein was diluted in carbonate buffer (pH=9.6) to an optimal concentration of 0.25 μg/ml for ELISA testing. The detailed procedure was described in our previous study (21 (link)). In brief, the diluted protein was coated onto the bottom of 96-well plates overnight at 4°C, followed by incubation using 2% bovine serum albumin (BSA) for 2 h at 37°C water baths. After washing with phosphate-buffered saline containing 0.05% Tween-20 (PBST), sera with the dilution of 1:100 or the dilution buffers without sera (blank control) were added into corresponding wells for incubation of 1 h at 37°C water baths. In this step, eight sera from four OC patients and four healthy controls were added into every plate for normalization among different plates (CV<15%). Then, plates were washed by PBST followed by incubating with HRP-conjugated goat anti-human IgG at 1:5m000 dilution for 1 h at 37°C water baths. A solution of 3,3’,5,5’-tetramethyl benzidine (TMB)-H₂O₂-urea was used as the detecting agent, and 2M sulfuric acid was added into each well as the stopping solution. The optical density (OD) was read at 450 and 620 nm by Multilabel Plate Reader (PerkinElmer).

The cell viability was determined by using the CyQUANT cell proliferation assay kit, which measures the total nucleic acid content. The cells were plated in 6-well plates at a seeding density of 2 × 10⁵ in 3 mL of culture medium containing 1% FBS. Then, the cells were treated with CB83, THC, CBD, and 5FU for 24 h at the respective IC₅₀ doses. The cells were harvested and were seeded (5000 cells/well) into a 96-well microplate in 200 µL of culture medium containing 1% FBS for 4 h. The medium was aspirated, and the plates frozen at −80 °C until used. The plates were thawed at room temperature and processed according to the manufacturer’s instructions. The analysis was carried out using a VICTOR Multilabel plate reader (excitation 485 nm/emission 520 nm; Perkin Elmer Victor 3V, Waltham, MA, USA).

After transfection, cells were seeded in 96-well plates (3 × 10⁴ cells/ml) with 100 ul medium in each well and cultured for 5 days. MTT assay was performed by adding 20 ul of MTT (5 mg/ml, AMRESCO, Solon, OH, USA) for 4 h at 37°C. Then, the formazan crystals were dissolved in DMSO (150 μl/well). The absorbance at 490 nm of each sample was measured using a multilabel plate reader (PerkinElmer). For the colony formation assay, 500 cells were seeded into 6-well plates and incubated at 37°C in a humidified atmosphere containing 5% CO2 in air for 10–14 days. Colonies were fixed with methanol, stained with 0.1% crystal violet and counted.

The effects of MA-DME, Black extract, and Transparent extract on cell viability were evaluated with an MTT assay in 96-well plates. HaCaT cells were seeded at a density of 1⋅10⁵ cells and incubated in 37 °C, 5% CO₂ conditions for 24 h. After that, various concentration of MA-DME (0 to 300 µg/mL) were treated and incubated for 24 h or 48 h. Then, 10 µL of EZ–cytox solution was added to each well, and the well plate was incubated for 4 h. Finally, measurement of absorbance at 450 nm wavelength was carried out utilizing a plate reader (Multi-label plate reader, PerkinElmer, Boston, MA, USA).

Assessment of elastase inhibition was conducted by the intensity of the solution color assay referring to the method of Tu and Tawata [36 (link)]. N-Succinyl-Ala-Ala-Ala-ρ-nitroanilide (AAAVN) elastase substrate was diluted with 0.1232 M Tris-HCl buffer solution (pH 8) to make a 1.0 mM concentration. Then, the elastase substrate was mixed with the 10 µL of sample in the 96-well plates and pre-incubated at 25 °C for 10 min. After pre-incubation, the reaction was initiated by adding 10 µL of elastase from porcine pancreas (7.5 units/mL) in Tris solution buffer to the pre-incubated mixtures. Finally, a microplate reader (Multi-label plate reader, PerkinElmer, Boston, MA, USA) was utilized to measure the absorbance at 410 nm.