Tc10 automatic cell counter

Growth rate analyses were initiated by seeding 24 well plates with 10⁵ cells per well and harvesting quadruplicates of these wells every 24 hr through to the 144 hr end-point. Growth curves were generated from live cell counts obtained with a TC10 Automatic Cell Counter (Bio-Rad) in the presence of Trypan Blue. Average specific growth rates [35 (link)]: μ, were obtained from the slopes of plots of ln(N_t/N_o) versus time, i.e., ln(N_t/N_o) = μt, where N_tis the number of cells at time ‘t’, N_o is the initial number of cells, and t is time. Consequently, the average length of the cell cycle was obtained from the equation: t_c = ln2/μ [35 (link)]. The rationale for the time intervals given in Table 1 is: The initial number of cells: N_o, can only be obtained after the cells have had time to settle, adhere, and begin to grow, i.e., 1 day after seeding the cells, as such, day 1 in Figure 4A equals day 0 = N_o Figure 4B. Thus, the first time interval of 48 hrs in Figure 4B means that the number of cells: N_t = N_o + (the cells that grew between 24 and 48 hrs), i.e., each time given on the x-axis of Figure 4B represents an interval of growth that begins at day 1 of the growth curves shown in Figure 4A. Hence, average specific growth rates in Table 1 bracket times; e.g., between 24 and 72 hrs, etc (Table 1).

CEM T cells were cultured in 96-well plates at 37°C and incubated with the indicated compound concentrations for 24 hrs. Cell viability was determined using trypan blue-based assay. The cells were supplemented with 0.2% trypan blue, transferred to a plastic disposable counting chamber and counted on a TC10 Automatic Cell Counter (Bio-Rad).
To assess cytotoxicity with calcein, media was removed and the cells washed with PBS in order to remove serum esterase activity that may cause an increase in fluorescence through the hydrolysis of calcein-AM. Cells were then supplemented with 0.2 μM calcein-AM (Molecular Probes, Invitrogen) for 10 min at 37°C. Fluorescence was measured using the luminescence spectrometer described above implementing 495 nm excitation and 515 nm emission filters.

PdNP cellular uptake was measured using ICP-AES (Agilent 720/730 spectrometer) analysis. HeLa (300,000 cells/well) and Caco-2 (1,800,000 cells/well) cells were seeded in a tissue culture-treated 6-well plate (EuroClone SPA, Milan, Italy) containing complete DMEM and incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 24 h. DMEM was then replaced with fresh medium containing 50 μg/mL of PdNPs and cells were incubated for 24 h. After washing cells three times with PBS without Ca²⁺ and Mg²⁺, they were harvested using trypsin-EDTA solution. Cell number was measured by a TC10 automatic cell counter (Bio-Rad). After a freeze/thaw lysis process, cell digestion was conducted overnight in CEM Discover SP Microwave synthesizer using fresh aqua regia. The solution was diluted 10 times with MilliQ water, and intracellular amount of Pd was then analyzed by ICP-AES. Each experiment was performed in triplicate.

The viability of BEAS-2B cells was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BEAS-2B cells were inoculated at a density of 5,000 cells per well in a 96-well plate and cultured for 24h. Cells were cultured with 10 μL MTT solution (5 mg / mL, Cat#: V13154, Life technologies, USA) at 37° C for 4h, then 200 μL of dimethyl sulfoxide was added to dissolve the crystals. Absorbance at 560 nm was read on a SpectraMax M2 microplate reader (Molecular Devices, CA, USA). As a complementary approach, cells were stained with trypan blue, total and alive cell numbers were measured with a TC10 automatic cell counter (Bio-Rad, CA, USA).

mirVana miR-483-3p inhibitors and mimics, and their respective negative controls (mirVana miRNA mimic and inhibitor negative control#1), were purchased from Thermo Fisher Scientific (Waltham, MA, USA). A total of 3 × 10⁶ GIST T1 and 882 cells were transfected with 50 pmol of miRNA mimics or inhibitors using Amaxa Nucleofector (Lonza, Basel, Switzerland), and program X01 and T20, respectively, according to the manufacturer’s instructions. For Western blotting, cells were harvested after 72 h of transfection.
For cell viability analysis, cells at 24 h post-transfection were treated with or without 1 μM imatinib for another 24 h, followed by staining with 0.4% trypan blue stain (Thermo Fisher Scientific, Waltham, MA, USA). Viable cells were counted using the TC10 automatic cell counter (Bio-Rad).