Scepter 2.0 automated cell counter

Inhibition of tumor cell growth was analyzed by quantification of cell numbers using Scepter 2.0 automated cell counter (EMD Millipore Billerica, Massachusetts, USA). The relative number of viable cells at the end of treatment was calculated as percentage of the untreated control.

For each MEF cell lines, cultures at 80–90% confluency were seeded in 6-well plates (CELLSTAR, Greiner Bio-One) at ∼6 × 10⁴ cells per well in 1.5 mL (∼6 × 10³ cell.cm⁻²) and incubated at 37°C and 5% CO₂. At each time point, culture medium was removed, cells were washed twice with phosphate buffer saline (PBS), detached with 0.5 mL Trypsin-EDTA (Gibco™, cat.no. 25200056) and resuspended in 1 mL culture medium (1.5 mL final cell suspension). Cell concentration and volume were measured directly after the sampling by diluting cell suspension four times in PBS and using Scepter™ 2.0 Automated Cell Counter with 60 μm sensors (Merck Millipore). Both total and viable cell count and volume were recorded.⁴⁷ The whole experiment was repeated three times with two technical replicates.

The concentration of cells in each sample (cells/ml) was estimated by counting cell number on a Scepter 2.0 Automated Cell Counter with 40 μM Scepter sensors (EMD Millipore; PHCC20040) in order to equalize cell numbers between biological replicates and between treatment groups prior to protein extraction. For cell population and growth analyses, initial cell counts (cells/mL) were determined manually using a hemocytometer and subsequently verified by sample analysis on the Scepter 2.0 Automated Cell Counter.

The MCF7 (ATCC, HTB-22), U87MG (ATCC, HTB-14), A549 (ATCC, CCL-185), and Caki-1 (ATCC, HTB-46) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and the MKN45 (JCRB Cell Bank, JCRB0254) cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan). All cell lines were tested for mycoplasma, cross-contamination, and genetic fingerprints. U87MG were cultured in Eagle’s Minimum Essential Medium (EMEM; ATCC, 30-2003, Manassas, VA, USA) containing 10% (v/v) fetal bovine serum (FBS; Gibco, 26140079, Carlsbad, CA, USA) and 1% (v/v) penicillin/streptomycin (Gibco, 15140122, Carlsbad, CA, USA). MCF7, MKN45, A549, and Caki1 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, 11875093, Carlsbad, CA, USA) containing 10% (v/v) FBS (Gibco, 26140079, Carlsbad, CA, USA) and 1% (v/v) penicillin/streptomycin (Gibco, 15140122, Carlsbad, CA, USA). All cells were cultured according to the manufacturer's manual and had been passaged for fewer than three months after thawing. Cell counting was performed with the Scepter™ 2.0 automated cell counter (Millipore, Billerica, MA, USA) and c-Met expression was confirmed by Western blot using cell lysates.

Microalgal growth, diameter, and volume were measured using a Scepter™ 2.0 Automated Cell Counter (Millipore, Berlington, MA, USA) every day of exposure. Briefly, 20 µL of cell culture was added to 2 mL of Coulter Isoton II diluent. The diatom concentration was expressed as the number of cells (10⁵)/mL of the medium, while the diameter and volume were expressed in µm and picoliters (pL), respectively. In addition, growth rates were calculated according to the following equation: $$\mathsf{\mu }=\ln (N_1/N_0)/(t_1-t_0)$$
where $N_1$ and $N_0$ represent cell concentrations at $t_1$ and $t_0$ [27 ].

P. tricornutum was purchased from the Culture Collection of Algae at Goettingen University (SAG) and was grown in the F/2 medium [26 ]. The stock culture was acclimated to the experimental conditions in a culture chamber at 16 °C, a light intensity of 40.5 µmol photons m⁻²s⁻¹, and a photoperiod of 12:12 light/dark. The stock solutions (1 mg/L) of BPAF and BPF were made in methanol, while the BPS was dissolved in distilled water. Stocks of microalgal cultures were inoculated in Erlenmeyer flasks containing 200 mL of F/2 medium and exposed in triplicate to the bisphenols BPAF, BPF, and BPS, as well as to their mixture. A concentration of 300 ng/L of each bisphenol or 100 ng/L of each of them in the mixture treatment was adopted. In addition, a control plus methanol was tested. Cells from the stock culture (inoculum) were added to obtain an initial concentration of 5 × 10⁵ cells/mL. The cell concentration was measured through a Scepter™ 2.0 Automated Cell Counter (Millipore, Berlington, MA, USA); the data had previously been validated using a Neubauer hemocytometer through a light microscope (Leica, Wetzlar, Germany). All the biochemical analyses were carried out in two different growth phases: exponential and at the beginning of the stationary phase, corresponding to 5 and 9 days of exposure, respectively.

Harvesting and transplantation BM cells were harvested in RPMI medium (Gibco, Life Technologies), and approximately 10⁷ cells were injected into the tail vein of recipient mice 30 min after MCAo [10 (link)]. Cell labelling A Vybrant^® CFDA SE Cell (CFSE) Tracer Kit (V12883, Invitrogen) was used for cell labeling and visualization in situ or by flow cytometry. Characterization BM cells harvested from IL-1Ra-Tg and LM mice (n = 5/group) were analyzed with regard to cell numbers and cell size using the Scepter 2.0 automated Cell Counter from Millipore. To assure compatibility with the Scepter correlation range (50 × 10⁵–1.5 × 10⁶ cells/ml), 10 µl of BM cell suspension was first counted using a Bürker-Türk counting chamber. Next, 100 µl of a BM cell suspension from IL-1Ra-Tg and LM mice were counted using the Scepter automated cell counter employing 40-μm Scepter sensors (Millipore). Histograms and overlays were computed utilizing the Scepter™ 2.0 Cell Counter and Software Pro system.