Giemsa s stain

The clonogenic assay was used to confirm the effectiveness of hTERT downregulation on the sensitization of cells to DOX, as previously described [36 (link)]. Briefly, the MCF7 and MDA-MB-231 cells were seeded into 60 mm plates in the concentration of 200 cells per well. After overnight incubation in standard conditions, on 21st-day post-transduction, cells were exposed to 10–500 nM of DOX. After 24 h, the medium was removed, and cells were washed twice with phosphate-buffered saline (PBS). Then the fresh medium was added, and cells were maintained for 14 days, with media change every four days. After that time, a 10 min fixation in methanol and staining with 1:20 aliquot of Giemsa’s stain (Sigma-Aldrich, St. Louis, MO, USA) for 1 h were performed. The wells were washed with distilled water, air-dried, and the colonies were enumerated. The experiment was repeated three times for each cell line [36 (link)].

Macrophages were plated on 12mm round glass coverslips (Bellco, US) placed in 24 well plates (Corning, UK) at a density of 4 x 10⁵ cells per well in RPMI-1640 media supplemented with 10% HiFCS. The plates were incubated at 37°C in 5% CO₂ for 24 hours. L. major stationary phase promastigotes were counted and dilutions of different concentrations of parasite (2 x 10⁵ to 6 x 10⁷) were pre-prepared in media to give initial parasite: macrophage ratios within the range of 0.5:1–15:; promastigotes were added to the macrophage cultures. The plates were placed in an incubator maintained at 34°C (temperature relevant for CL [14 (link)]) and 5% CO₂ for 24 hours. Subsequently, two thirds of the glass coverslips were transferred to the media perfusion system and maintained under flow conditions at a flow speed of 360 μl/min for 72 hours in a 34°C, 5% CO₂ incubator. The remaining coverslips were used for the static control, with macrophages maintained in the same culture medium without flow. The cells were methanol (Sigma, UK) fixed and stained with Giemsa’s stain (Sigma, UK). The infection rate of the macrophages was assessed visually using an oil immersion microscope (100x magnification Zeiss, UK) by counting the number of infected cells per 100 macrophages. Values for percentage infection throughout are shown as mean ± standard deviation.

Cell migration was evaluated using Transwell inserts (6.5 mm diameter) (Corning Costar) in 24 well plates. Pre-treated THP cells (50,000 cells/well) were added to the upper chamber of the insert. The lower chamber contained 1 mL of normal glucose (NG) or high glucose (HG) (20 mM) RPMI/1% human albumin with or without cyclophilin A as chemokine. LPS (10 µg/mL) was used as positive control throughout the experiments. The plates were incubated for 4–24 h and stained with Giemsa’s stain (Merck Chemicals). The number of cells appearing on the lower face of the filter was recorded in three random fields for each well. The chemotactic index (CI) was calculated as the number of cells migrating towards the test sample divided by the number of cells migrating towards the control medium using light microscopy. The number of cells migrating to the bottom chamber was counted by flow cytometry. The inserts were fixed with 4% para-formaldehyde (PFA) and stained with Giemsa’s stain. The cells migrating to the reverse surface of the membrane were counted by light microscopy at 20× magnification in four different random fields. After migration the cells in lower chamber were counted and analyzed directly by flow cytometry (Becton–Dickinson).