Scepter cell counter

The human ovarian carcinoma cells, ES-2 and SKOV-3, were seeded onto 96-well microplates with 5 × 10³ cells per well and were incubated overnight. The following day, cells were treated with 0 to 1000 μg/mL of FWGE for 24 h or 48 h. The cell viability of the treated cells was determined using a Scepter cell counter (Merck Millipore Billerica, MA, USA) based on cell size measurements [25 (link)]. A morphological observation was performed using a Nikon Eclipse TS100 optical microscope (Nikon Instruments, Melville, NY, USA), and the observation was photographed at 100x magnification.

HRMECs were cultured in endothelial cell medium supplemented with endothelial cell growth supplement (ECGS), 5% fetal bovine serum (FBS), and 1% antibiotic–antimycotic. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂. The media were changed every other day until cells reached confluency. For seeding, cells were trypsinized and counted using a Scepter Cell Counter (Merck Millipore Co., Burlington, MA, USA).

On the 10th day of proliferation, cells were trypsinized. The next step was counting the cells using a Scepter Cell Counter (Merck Millipore, Darmstadt, Germany) and transferring 30,000 cells/well to Cellware 6-well plate covered by Collagen I (Greiner Bio-One, Monroe, NC, USA). When the cells obtained 80% confluence, the proliferation medium was replaced by differentiation medium (2% HS/ Dulbecco’s Modified Eagle Medium (DMEM)/AB). After the 2nd day of differentiation, 0.125 µM of GO (TCI Chemicals, Portland, USA), dissolved in 0.04 µL/mL DMSO as a vehicle, which was also used in the control medium, was added for 24 h. The MTT assay was used to choose the concentration of GO and H₂O₂ [25 (link)]. During the last part of the experiment (the last one hour of GO incubation), H₂O₂ (3 mM, Sigma Aldrich, Poznań, Poland) was added. The main purpose of H₂O₂ administration was to cause cell damage (Figure 1).

Cryopreserved primary human astrocytes were purchased from Innoprot (P10251, P10254). After thawing, cells were grown in cell culture flasks for 4 days and then plated on glass coverslips coated with poly-d-lysine (diameter 22 mm; Sigma-Aldrich, P6407) at a cell density of 1.5 × 10⁴ cells per coverslip (transfection and immunocytochemistry experiments) or on 12-well culture plates (TPP, 92112) at a cell density of 3 × 10⁴ cells per well (RT-PCR experiments) as measured by a Scepter cell counter (Merck KGaA, Darmstadt, Germany). Cell cultures were maintained in astrocyte growth medium (high glucose Dulbecco’s modified Eagle’s medium [Sigma-Aldrich, D5671] supplemented with 10% fetal bovine serum [FBS; Sigma-Aldrich, F7524], 1 mM sodium pyruvate [Sigma-Aldrich, S8636], 2 mM l-glutamine [Sigma-Aldrich, G3126], 5 U/ml penicillin, and 5 µg/ml streptomycin [Sigma-Aldrich, P0781]) at 37 °C, 5% CO₂ atmosphere, and 95% relative humidity. Cells were used in experiments approximately 24 h after plating on coverslips/culture plates.

Hep3B and HepJ5 cells were plated onto 96-well plates, with 5 × 10³ cells per well, and cultured overnight before treatment. To evaluate the antitumor effects of AE-SN, the cells were treated with 0 to 2.0 mg/mL of AE-SN for 48 h. To evaluate the antitumor effects of integrated treatment with the chemotherapeutic drugs and AE-SN, the cells were treated with 0 to 20 μM cisplatin or 0 to 10 μM and 0, 0.5, or 1.0 mg/mL of AE-SN for 48 h. In this study, cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table 1).
Two approaches, namely, microscopic observation and measurement of the cell size distribution, were performed to inspect the morphological changes in AE-SN-treated HCC cells. General morphological changes were observed using a Nikon Eclipse TS100 optical microscope (Nikon Instruments, Melville, NY, USA) and photographed at 100x magnification, whereas the distribution of cell diameter was measured using a Scepter cell counter (Merck Millipore Billerica, MA, USA), which divided surviving cells from cell fragments and debris in a borderline of 12 μm [18 (link)].