Gentamycin sulphate

In order to quantify the internalised bacteria after a 3 h invasion, invasion assays were carried out as described previously^{23 (link)} with the following alterations. After 3 h, the media overlying the infected HT29 cells was changed to complete McCoy’s 5A medium supplemented with 200 μg/ml gentamycin sulphate (Lonza) and infected HT29 cells were incubated at 37 °C under microaerophilic conditions for a further 2 h. After this, cells were washed 3 times with PBS and lysed with 1% Saponin in PBS for 15 min. Serial dilutions of the cell lysates were carried out using MH broth and plated on MH agar. All MH plates were incubated for 48 h under microaerophilic conditions using Campygen gas packs (Oxoid) at 37 °C followed by counting of colony forming units.

Human embryonic kidney 293 cells (HEK293A) (R70507, Thermo Fisher Scientific, Waltham, USA), human embryonic lung fibroblasts (IMR-90) (ATCC CCL-186, LGC Standards, Germany) and primary murine cardiac fibroblasts (isolated from mouse hearts) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Gibco, Cat # 41965-062, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, Cat # 16000-044, USA) and 50 µg/ml gentamycin sulphate (Sanofi, Cat # 453130, Norway). Human Umbilical Vein Endothelial Cells (HUVEC) (ATCC CRL-1730, LGC Standards, Germany) were maintained in Endothelial cell Basal Medium (EBM) (Lonza, Cat # CC-3121, Switzerland), including the EGM SingleQuots growth supplements (Lonza, Cat # CC-4133, Switzerland) and 50 µg/ml gentamycin sulphate.

Cell culture and treatment OVcar3, SKov3 epithelial ovarian cancer cells were maintained in RPMI 1640 (Lonza) supplemented with 10% v/v foetal bovine serum, 50 µM gentamycin sulphate and 2 mM L-glutamine (Lonza). Cells were routinely incubated at 37 °C in 5% CO2-95% air atmosphere and passaged twice every 6 days. Ovarian cancer cells (1×10⁵ cells) were plated in 6-well plates for use in flow cytometry analysis. When immunohistochemistry was utilized, cells were cultures in wells containing sterile cover slips to 80% confluence. The following day, cells were treated with varying concentrations of doxorubicin—DX (Sigma) alone (2.5, 6.3, 12.5, 25 µM). Separately, cells were treated with varying concentrations of thapsigargin -TG (0.2, 0.5 µM) with and without 2.5 µM DX for 16 h. All cells were then exposed to 2 µg/ml (final concentration) FITC-CRT for 30 min and washed once in sterile PBS. Next, for flow cytometry analysis, cells were removed from wells of plates by trypsinizing them with 0.4 mls of trypsin—EDTA—0.05% at 37 °C for 5 min before adding 1 ml of DPBS (Dulbecco’s Phosphate Buffered Saline—Fisher) without calcium, magnesium to neutralize the trypsin. Then, the cells were centrifuged at 1500 rpm for 5 min to obtain a pellet. The DPBS was removed and the cells resuspended in 1 ml of fresh DPBS.