24 well cell culture plate

Cell growth was determined using cell counting assay, in which 2.0 × 10⁵ cells/well were seeded in a 24-well cell culture plate (SPL Life Science, Pocheon, Republic of Korea) on day zero and counted every 24 h using a hemocytometer (Marienfeld, Lauda-Königshofen, Germany) under a phase-contrast microscope (Olympus CKX41; Olympus Corporation, Tokyo, Japan).

Spores from 9-day-old PDA cultures were collected in PDB, filtered through two layers of Miracloth, and diluted to 5 × 10⁴/ml, and 30 μl of spore suspension was added to each well of a 24-well cell culture plate (SPL Life Sciences, South Korea). The plate was incubated at 22°C, and germination rates were determined at 2-h intervals using an inverted microscope (Olympus). Experiments included four replications with 50 ± 10 spores per sample. The experiments were repeated three times.

Anthracene (99% purity, CAS No. 120–12–7) was purchased from Sigma Aldrich (Saint Louis, MO, USA) and test solutions at the desired concentrations were prepared by serial dilution from stocks in high-performance liquid chromatography (HPLC)-grade dimethyl sulphoxide (DMSO; Sigma Aldrich). Microplate toxicity tests of 96 h in duration were conducted in 24-well cell culture plates (well diameter = 15.6 mm, growth area = 1.9 cm²; SPL Life Sciences, Gyeonggi-Do, Korea) with a test volume of 2 mL per well. Equal volumes of cell suspension and Anthracene stock solutions were mixed to obtain final concentrations of 0.625, 1.25, 2.5, 5, 10 and 15 mg L⁻¹, along with untreated controls. The concentration of the carrier solvent did not exceed 0.2% v/v of the test culture volume. The initial cell density was 10 ± 0.5 × 10⁴ cells mL⁻¹ of suspension. An additional solvent toxicity test (96 h) was conducted with a maximum DMSO concentration of 0.2% v/v. Organisms were exposed to nominal concentrations of Anthracene and all treatments were performed in triplicate. The well plates were covered with parafilm to avoid evaporation and mixing of the volatile toxicant.

HeLa cells were cultured into 24-well cell culture plates (1 × 10⁵ cells/well) overnight (SPL Life Sciences Co., Ltd., Gyeonggi-do, Korea) and were washed with serum-free DMEM. Bacterial cells were then infected to the HeLa cells at an MOI of 20 and then incubated in a 5% CO₂ incubator at 37°C for 120 min. V. vulnificus culture suspensions were 10-fold serially diluted with free DMEM, and then the serial dilutions (10 μL) were loaded on HI agar plates overnight.

Cell viability was assessed by a methyl-thiazoldiphenyl-tetrazolium (MTT) assay (MTT Cell Growth Assay Kit, Chemicon, Rosemont, IL, USA) [19 (link),21 (link),22 (link)]. We seeded hDPSCs at a density of 1.0 × 10⁴ cells/well on 24-well cell culture plates (SPL Life Sciences, Pocheon, Korea). The material disks were placed into inserts with a 0.4 μm pore size (SPLInsert; SPL Life Sciences), and these inserts were maintained over the attached hDPSCs for 5 days. Then, the hDPSCs were treated with MTT solution according to a previously reported method at 0, 1, 2, 3, and 5 days [19 (link)]. We measured the absorbance at 570 nm. Positive controls were hDPSCs cultured without experimental disks. Each group was evaluated in quadruplicate.

A Cell Counting Kit test (CCK-8) (CK04-13; Dojindo, Kumamoto, Japan) was used to evaluate the cytotoxic effects of the three kinds of experimental disks. The cell proliferation rate of hBMSCs was analyzed shortly after incubation, and analyzed again 1, 2, and 4 days after incubation. hBMSCs were cultured at a density of 1.0 × 10⁴ cells/well in 24-well cell culture plates (SPL Life Sciences, Pocheon, Korea) in growth medium. After 24 h of incubation of the adherent cells, the optical density value was obtained.
Individual disks were placed on an insert (SPLInsert; SPL Life Sciences) with pores 0.4 mm in size, while the inserts were located above the hBMSCs. Each well was supplemented with 1 mL of additional medium in order to cover the top of the disk. hBMSCs cultured without experimental disks were used as a control group. Emdogain at the initial concentration of 30 mg/mL was added to groups 2, 4, 6, and 8, and diluted in α-MEM for experimental use at a final concentration of 100 μg/mL. A total of 20 μL CCK-8 solution was added to each well, and the plates were placed in an incubator at 37 °C for 1 h. Later, we rinsed each well with PBS, and added dimethyl sulfoxide for dissolving the synthesized formazam. Absorption at 450 nm was measured using an absorbance microplate reader (Power Wave XS; BioTek Instruments, Winoski, VT, USA). Each group was measured in octuplicate.

Saliva was collected from healthy individuals (n = 3, pooled saliva, aged between 20 and 30 years, non-smokers) with no dental decay or gum disease, and those who had not taken antibiotics in the last 3 months. These individuals were asked to avoid all oral hygiene activities for 24 h before donating their saliva. The collected saliva was filtered using sterilized glass wool following stimulation (Duksan Chemicals, Ansan, Republic of Korea). This filtered saliva (1.5 mL) was applied to HA disk specimens arranged in 24-well cell culture plates (SPL Life Sciences, Pocheon, Republic of Korea). The plates were incubated at 37 °C in an atmosphere with 10% CO₂ (BB15 CO₂ incubator; Thermo Fisher Scientific, Waltham, MA, USA) for 4 h. Post incubation, the saliva was removed from the HA disk specimens, and a fresh medium (final pH 7.0) containing 0.5% sucrose (0.1 mL) and basal medium mucin (1.4 mL) was added to each specimen. This medium was subsequently replaced daily for either 2 or 6 days, allowing for the development of oral microcosm biofilms under the same temperature and CO₂ conditions. According to the findings of a preliminary experiment regarding the effect of biofilm thickness on the penetration of QLF-D’s blue light, biofilm thickness was adjusted to 1 mm, which did not impede light penetration.