Casy model tt

To characterize growth behaviour of the novel established A375‐hS100A4 cell line in vitro and in vivo experiments were performed. Therefore, A375 and A375‐hS100A4 cells were seeded at a density of 1 × 10⁵ per well in a 6‐well plate and cultured for 5 days. Proliferative growth in vitro was estimated by counting the total number of living cells using a Casy Model TT cell counter (Roche, Mannheim, Germany). Moreover, both wild‐type A375 and transfected A375‐hS100A4 cells were used in a pilot experiment to establish melanoma xenograft models in NMRI (nu/nu) mice using a protocol published elsewhere with some modifications 20. In brief, single‐cell suspensions of both cell lines each with 5 × 10⁶ cells in 100 μl phosphate‐buffered saline were subcutaneously injected into the right upper flank of the mice. Lengths and widths of the tumours were measured twice a week using a sliding caliper. Tumour volumes were calculated by using the formula π6 × length × width². Mice were killed at day 23. Animal experiments were carried out according to the guidelines of the German Regulations for Animal Welfare. The protocol was approved by the local Ethical Committee for Animal Experiments (reference number 24‐9168.11‐4/2012‐1).

Cell diameter was estimated for cells growing in YPD to the early exponential growth phase. The cell size was measured before and 20 s after the addition of NaCl (final concentration 1 M) to the sample. A cell counter (CASY model TT; Roche Innovatis AG) with a 60 μm capillary was used. The experiment was repeated four times, each time 3–5 × 10⁴ cells were analyzed for each strain and each set of conditions. Intervals containing the most typical 60% of the cell population were visualized using a box plot diagram with the mean diameter from the observed interval (2.5–9 μm) inside the box. Average results are shown ± SD.

A total of 1 × 10⁵ cells were seeded onto 6-well plates at day 0. Starting at day 1, we counted the cells every 48 hrs until day 9 using the CASY Model TT cell counting device (Roche Innovatis AG, Basel, Switzerland). Each experiment was performed in duplicate and repeated three times.

For doubling
time characterization
in mutants and WT parasites, the culture density was adjusted to 1
× 10⁶ promastigotes/mL in M199. After an incubation
for 24 h at 28 °C, the cell culture density was determined using
a cell counter (CASY model TT, Roche Diagnostics) with a 60 μm
capillary and exclusion of particles with a pseudo diameter below
2.0 μm. The cell density was adjusted again to 1 × 10⁶ promastigotes/mL in a new flask. This procedure was repeated
for 4 d. The doubling time (DT) was calculated using the following
formula.

The estrogen-sensitive human breast adenocarcinoma cell line MCF-7 and the estrogen-independent adenocarcinoma cell line MDA-MB-231 were obtained from CLS Cell Lines Service (Eppelheim, Germany). All cells were routinely cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 1% penicillin-streptomycin. Cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere. Both cell lines were maintained as monolayers in 75 cm² plastic flasks (TPP, Trasadingen, Switzerland). After reaching confluence, the cells were washed twice with 3 mL phosphate buffer saline (1× PBS) and incubated with 3 mL 0.05% trypsin/0.02% EDTA for 1 min. The trypsin treatment was halted by adding complete media, and the cells were centrifuged (230× g, 3 min) and resuspended in complete medium. The cell number and viability were determined using the Casy Model TT (Roche Life Sciences, Indianapolis, IN, USA).