Casy model tt cell counter

Promastigote form L. mexicana (WHO strain MNYC/BZ/62/M379), L. major Friedlin and their genetically modified derivatives were grown at 28°C in M199 medium (Life Technologies) supplemented with 2.2 g l⁻¹ NaHCO₃, 0.005% haemin, 40 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid pH 7.4 and 10% FCS. Relevant selection drugs (Melford Laboratories Ltd.) were added to the medium at the following concentrations: 32 µg ml⁻¹ Hygromycin B, 20 µg ml⁻¹ Puromycin Dihydrochloride, 5 µg ml⁻¹ Blasticidin S Hydrochloride, 40 µg ml⁻¹ G-418 Disulfate, 50 µg ml⁻¹ Nourseothricin Sulfate and 25 µg ml⁻¹ Phleomycin. Trypanosoma brucei SmOx B4 [49 (link)] was grown in HMI-9 [54 (link)] at 37°C in 5% CO₂, T. brucei SmOx P9 in SDM-79 [55 (link)] at 28°C. Relevant selection drugs (Melford Laboratories Ltd.) were added to the medium at the following concentrations: 0.2 µg ml⁻¹ Puromycin Dihydrochloride, 5 µg ml⁻¹ Blasticidin S Hydrochloride, 5 µg ml⁻¹ G-418 Disulfate, 5 µg/ml Hygromycin B. Cell culture densities were measured using a CASY model TT cell counter (Roche Diagnostics) with a 60 µm capillary and exclusion of particles with a pseudo diameter below 2.0 µm.

The murine leukemic cancer cell line L1210 was obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). We used three variants of the mouse lymphocytic leukemia cell line L1210: (i) ABCB1-negative drug-sensitive parental L1210 cells (ACC-123, S); (ii) ABCB1-positive, drug-resistant cells (R) overexpressing ABCB1 due to selection with vincristine [24 (link)]; and (iii) ABCB1-positive, drug-resistant cells (T) overexpressing ABCB1 due to stable transfection with Addgene plasmid 10957 (pHaMDRwt), and a retrovirus encoding full-length ABCB1 cDNA [72 (link)]. Transfection and cell characterization were completed as described elsewhere [25 (link)]. Cells were incubated in RPMI 1640 medium with L-glutamine (1 mg/mL) supplemented with 4% fetal bovine serum and 1 μg/mL gentamycin (all purchased from Gibco, Langley, OK, USA), at 37 °C, in a humidified atmosphere with 5% CO₂. Cell viability was monitored by using a CASY Model TT-Cell Counter (Roche Applied Sciences, Madison, WI, USA). All cell variants (S, R and T) were cultivated in the absence or presence of SFN at an appropriate concentration and were used for further examination.

The cells (S, R, and T; inoculums: 1 × 10⁶ cells) were cultured in 4 mL RPMI 1640 media with L-glutamine (1 μg/mL), 8% fetal bovine serum, and 1 μg/mL gentamycin (all purchased from Gibco, Langley, OK, USA) in a humidified atmosphere with 5% CO2 at 37 °C for 48 h in the absence or presence of tunicamycin (0.1 μM). This procedure was termed passaging and was repeated three times. The number of viable cells after each passage was counted using a CASY Model TT Cell Counter (Roche Applied Sciences, Madison, WI, USA). R cells were cultured for two passages without vincristine (VCR) prior to the experiments.

The following three L1210 cell variants were used in this study: (i) S-drug-sensitive parental cells obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) ACC-123; (ii) R—P-gp-positive drug-resistant cells that overexpress P-gp after selection with vincristine [44 (link)] obtained from Gedeon Richter Co., (Budapest, Hungary); and (iii) T—P-gp-positive drug-resistant cells that overexpress P-gp following stable transfection with the P-gp gene [15 (link)], using the Addgene plasmid 10957 (pHaMDRwt), a retrovirus encoding the full-length P-gp cDNA [45 (link)]. The cells (S, R and T; inoculums 1 × 10⁶ cells) were cultured in 4 cm³ RPMI 1640 media with l-glutamine (1 mg cm⁻³), 4% fetal bovine serum and 1 μg cm⁻³ gentamycin (all purchased from Gibco, Langley, OK, USA) in a humidified atmosphere with 5% CO₂ and air at 37 °C for 48 h in the absence or presence of either tunicamycin (0.1 μmol·dm⁻³) or GalNAc-α-O-benzyl (0.1 mmol·dm⁻³). This procedure was termed as passage and was repeated three times. Numbers of viable cells after each passage were counted using a CASY Model TT Cell Counter (Roche Applied Sciences, Madison, WI, USA). R cells were cultured for two passages without VCR prior to the experiments.

Cells (2 × 10⁴ to 3 × 10⁴ cells/well depending on the cell lines) were plated in 24-well plates in their regular medium. The next day, after being washed with PBS, cells were transferred to -SG medium or CM and treated with 10 µM PH755 (RAZE Therapeutics) diluted in DMSO or DMSO alone. For the counting step, cells were trypsinized, suspended in PBS-EDTA and counted with a CASY Model TT Cell Counter (Innovatis, Roche Applied Science). Relative cell number at each time point was calculated based on the number of cells measured before the medium change. For the growth curve experiment with formate and glycine supplementation, HT-29, HCT116 and DLD-1 cells were seeded in 24-well plates (2 × 10⁴ cells/well). Sodium formate (Fluka Analytical, 71540) (1 mM) and/or glycine (0.4 mM) were diluted in -SG medium + 10 µM PH755 and medium was refreshed every two days.

Variants of L1210 cells (10⁵ cells per well) were plated/seeded in 6-well culture plates (time 0) and cultured in a humidified atmosphere with 5% CO₂ in air at 37 °C. The number of viable cells was detected in a CASY Model TT Cell Counter (Roche Applied Sciences, Madison, WI, USA) in triplicate at different time intervals between 0 and 48 h. The time course of cell proliferation over this time interval followed first-order kinetics, i.e., according to Equation (2): $\mathrm{N}=\mathrm{I}\times {10}^{\mathrm{k}\times \mathrm{t}}$
where N is the number of cells after cultivation for time t, I is the number of cells in inoculum and k is the first-order kinetic constant.