Six well cell culture plates

HeLa cells, HeLa Kyoto cells, Chinese hamster ovary (CHO-K1) cells and human embryonic kidney 293 cells were maintained in DMEM medium with 4.5 g l⁻¹ glucose (Gibco), supplied with 10% (v/v) FCS (Gibco) in T75 cell culture flasks (Greiner). Every 2–3 days, cells were passaged using PBS (Sigma-Aldrich) and 0.5% trypsin/0.02% EDTA/PBS (GE Healthcare). All cell lines were cultivated in a humidified tissue culture incubator at 37 °C and 5% CO₂. Mycoplasma contamination tests were carried out regularly, following the guidelines described24 . Transient transfection was performed with Lipofectamine 2000 (Life Technologies), following the manufacturer's instructions. For fixation and staining, 2 × 10⁴ cells per well were seeded into eight wells on cover glass II slides (Sarstedt) and transfected with 0.2 μg DNA per well. For squeezing experiments, 8 × 10⁵ cells were seeded into six-well cell culture plates (Greiner) and transfected with 2 μg DNA per well. After transfection, cells were incubated 12–48 h at 37 °C and 5% CO₂ until experiments were performed.

Cells were grown on six-well cell culture plates (Greiner Bio One Ltd., Stonehouse, UK). Post-treatment with PMA both U-937 and HL-60 cells were washed with PBS and membrane integrity was monitored under tested experimental conditions by staining cells with amphiphilic styryl dye FM4-64 (15 μM, 5 min, RT). FM4-64 was visualized in cells placed on slides by CLSM using excitation by a 543 nm He-Ne laser and 655–755 nm emission filter. Imaging was combined with a transmitted light detection module with 405 nm diode laser excitation and Nomarski/differential interference contrast (DIC) filters to follow morphology of low-contrast cells.

The growth inhibition assay with L. minor was performed according to a modified version of the test protocol described in detail by Drost et al. (2007 (link)). The plants were grown in open Erlenmeyer flasks in sterilized Steinberg medium (pH 5.5 ± 0.2) in a climate chamber with a constant temperature of 25 ± 2 °C. To exclude pH effects on plant growth, the pH was checked at the beginning and end of the test. Based on control sample evaluation, the pH changes did not affect growth inhibition. The chamber was illuminated continuously with a maximum of 6 klx. The assays were performed on six-well cell culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany). The tests were performed using six different concentrations in three replicates of every compound and each test was repeated three times. Six organic solvent free controls (containing only medium) and six solvent controls (containing 0.2 % DMSO in medium) were used in each test. The test started with one plant consisting of three duckweed fronds, and the measured endpoint was the inhibition of the growth rate determined by the frond area (mm²), which was calculated for the treated plants in relation to the untreated controls. The frond area was detected using a Scanalyzer from Lemnatec GmbH (Würselen, Germany).