Z2 cell counter

Cell lines for the investigation were generated from the tsetse-transmissible T. b. brucei AnTat 1.1 (wild-type or WT) cell line [22 (link)]. Procyclic trypomastigotes of the WT cell line and its derivatives were cultured in SDM-79 [23 (link)] supplemented with 10% foetal bovine serum and 20 mM glycerol [24 (link)](SDMG for SDM-79 + Glycerol). Cell concentration was determined using the Z2 cell counter (Beckman Coulter). Bloodstream AnTat 1.1 trypanosomes were cultured in Creek’s minimum medium [25 (link)] at 37°C, 5% CO₂ and allowed to attain 2 X 10⁶ parasites/ml culture prior to harvest for transformation to the procyclic stage, IFA or western blots for the comparison of AK3 amounts between bloodstream and procyclic trypanosomes. For transformation, bloodstream trypanosomes were suspended in DTM medium with 3 mM isocitrate/cis-aconitate and cultured at 27°C for 24 h [26 (link)]. Transformed parasites were subsequently passaged into SDMG medium. Drug selection was always removed prior to generating growth curves. Cells were spun down, resuspended in medium without drugs prior to measuring growth over time. Cultures were maintained in medium without drugs throughout the duration of the experiment. The only exception for this was for the inducible cell line, where tetracycline was added to the induced culture after re-suspension in medium.

The wildtype PF T. brucei Lister 427 strain and tet-inducible PF 29–13 cells were grown in vitro at 27°C in SDM-79 medium containing hemin (7.5 mg/ml) and 10% fetal bovine serum (FBS) [32 (link)]. Meanwhile, the following cultures were maintained at 37°C with 5% CO₂ in HMI-9 media containing 10% FBS: wildtype BF T. brucei Lister 427, the derived BF single marker (SM) strain [32 (link)], laboratory induced dyskinetoplastic T. brucei Dk164 [33 (link)] and genetically modified Dk T. b. evansi cells [34 (link)]. The PF 29–13, BF SM and T. b. evansi cell lines constitutively express the ectopic bacteriophage T7 RNA polymerase and the prokaryotic tet repressor, which allows for the tet inducible expression of either ectopic V5-tagged proteins or dsRNA. As described previously [32 (link)], the appropriate cells were then transfected with NotI linearized pZJM or pT7_V5 plasmids containing the TbIF1 gene. Both plasmids were targeted to the rDNA intergenic spacer region. The addition of 1 μg/ml of tet into the media triggers either the induction of RNAi or the expression of tagged TbIF1. Throughout the analyses, a Z2 Cell Counter (Beckman Coulter Inc.) was used to measure cell densities in order to maintain the cultures within their exponential mid-log growth phase of 1x10⁶ to 1x10⁷ cells/ml for PF and between 1x10⁵ to 1x10⁶ cells/ml for BF cells.

Stock solutions for test compounds were prepared at 10 mM in DMSO. To measure the antileishmanial activity of these inhibitors, log phase promastigotes were inoculated in complete M199 media at 2.0 × 10⁵ cells/ml in 24-well plates (1 ml/well). Inhibitors were added to various concentrations and control wells contained DMSO only (0.1–0.5%). Culture densities were determined after 48 h using a Beckman Z2 Cell Counter.

L. donovani promastigotes were cultivated in complete M199 media (pH 7.4) at 27 °C until they reach the stationary phase. For heat tolerance, promastigotes were incubated at 37 °C. To test their sensitivity to acidic pH, promastigotes were transferred to a pH 5.0 medium (same as the complete M199 medium except that the pH was adjusted to 5.0 using hydrochloric acid). For starvation response, promastigotes were transferred to PBS (pH 7.4). For resistance to oxidative or nitrosative stress, parasites were incubated in various concentrations of H₂O₂ or S-nitroso-N-acetylpenicillamine (SNAP). Cell viability was determined at the indicated times by flow cytometry after staining with 5 μg/ml of propidium iodide. Parasite growth was monitored using a Beckman Z2 Cell Counter.

Human colon cancer HT29 [33 (link)], Ls-174-T [34 (link)] and LoVo [35 (link)], glioblastoma U251 [36 (link)] and T98G [37 (link)] cell lines were employed. Cells were seeded in a 12-well plate and maintained at standard conditions (37 °C in a humidified 5% CO₂ atmosphere) for 24 h, then treated and incubated for additional 48 h. Following 48 h of treatment, cells were detached from plates with trypsin and counted using a BECKMAN COULTER Z2 cell counter (Beckman, Pasadena, CA, USA). Then, the IC₅₀ values for each compound were calculated. The IC₅₀ represents the 50% inhibitory concentration, which is defined as the compound concentration inhibiting cell proliferation of 50% [7 (link)]. The reported IC₅₀ values are average values (±standard deviation) relative to three independent experiments.

FITC (fluorescein isothiocyanate) annexin V (apoptosis marker) and propidium iodide (PI, necrosis marker) stainings were carried out according to the manufacturer's instructions (FITC Annexin V Apoptosis Detection Kit I, 556547, BD Pharmingen, Heidelberg, Germany), followed by FACS (fluorescence-activated cell sorting, BD FACSCalibur, Heidelberg, Germany). Membrane-adherent hPDL fibroblasts were washed twice with PBS (phosphate-buffered saline) of 4°C and immediately resuspended at 10⁶ cells/pro ml (Z2 cell counter, Beckman Coulter, Krefeld, Germany) in 10x binding buffer (0.1 M HEPES/NaOH pH7.4, 1.4 M NaCl, 25 mM CaCl₂). 100 μl was then transferred to a 5 ml FACS tube, and 5 μl FITC annexin V and 5 μl PI were added. hPDL fibroblasts were gently vortexed and incubated in the dark at room temperature for 15 min. Prior to flow cytometry, 400 μl binding buffer was added to each tube (FSC: 5 V, SSC: 349 V, FITC: 350 V, PerCP-Cy5-5: and 450 V; Threshold FSC: 5000; Laser Delay Blue: 0.00, Red: 24.10; Area scaling Blue: 1.79, Red: 1.80; FSC Area Scaling: 1.08).