Phosphate buffer saline (pbs)

Quantification of the colibactin-associated genotoxic effect by megalocytosis assay was performed as previously described [23 ]. Briefly, E. coli NC101 and K-12 strains from glycerol stocks were grown in LB at 37 °C, shaking overnight. Strains were sub-cultured in EMEM for 4 h. Caco-2 cells were dispensed (1 × 10⁵ cells/well) in a 24 well tissue culture plate (Falcon) at 37 °C in a 5% CO₂ atmosphere. After 24 h, Caco-2 cells were infected at a multiplicity of infection (MOI) of 50 with indicated E. coli strains. After 4 h of infection, the cells were washed at least three times with phosphate buffer saline (PBS) (Wisent Inc) and incubated for 72 h in cell culture medium supplemented with 200 μg/ml gentamicin (VWR). Cells were fixed with 4% paraformaldehyde (Thermo Fisher) for 15 min, washed and stained with 1 mM methylene blue (Sigma Aldrich). Pictures were taken under a Nikon Eclipse TE300 microscope (Nikon Healthcare, Québec, Canada) and images were acquired using the NIS-Elements BR4.00.03 software (200X magnification). methylene blue extraction solution was used to quantify cell damage and megalocytosis at 660 nm absorbance (Spark® multimode microplate reader).

ZIKV stocks were prepared by passaging in Vero cells. Briefly, 6.0 × 10⁶ Vero cells were plated in a T182.5 flask. On day 1, the growth medium was removed and cells were washed with Phosphate Buffer Saline (PBS) (Wisent, St Bruno, QC, Canada). Cells were then infected at a multiplicity of infection (MOI) of 0.5 in 10 mL of Eagle’s minimal essential medium (EMEM) (Wisent, St Bruno, QC, Canada) and incubated (37 °C, 5% CO₂) for 2 h. The infection medium was then removed and replaced with ZIKV infection medium: DMEM supplemented with 2% FBS; 1% non-essential amino acids; 1% L-glutamine; 50 U/mL Penicillin and 50 μg/mL Streptomycin (Wisent, St Bruno, QC, Canada), and; 15 mM Hepes buffer (Sigma-Aldrich, Oakville, ON, Canada). At two days post-infection, the supernatant was filtered through a 0.45 μM membrane, viral stocks were tittered by plaque forming unit (PFU) assay, aliquoted and stored at −80 °C.

SK-LMS-1 Human leiomyosarcoma cell line was procured from ATCC (HTB-88, ATCC, Manassas, VA, USA). STS117 Human soft-tissue-sarcoma (STS) primary cell line harboring a loss of function mutation TP53 was derived from patients’ primary extremity STS diagnosed as an undifferentiated pleomorphic sarcoma. STS117 cell line was kindly provided by Dr. R. Gladdy (Mount Sinai Hospital, Toronto, ON, Canada) [17 (link)]. SK-LMS-1 was cultured in EMEM (Wisent Inc., St-Bruno, QC, Canada), STS117 was cultured in DMEM F12 (Wisent Inc.), both supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Thermo Fisher Scientific, Saint-Laurent, QC, Canada) and 1% Penicillin–Streptomycin solution (Wisent Inc.). SK-LMS-1 and STS117 cells were maintained by subculturing at 80% confluency. Briefly, the medium was aspirated, cells were washed with Phosphate Buffer Saline (PBS) (Wisent Inc.) and were trypsinized with 0.025% trypsin EDTA (Wisent Inc.) for 2–3 min at 37 °C. Once cells are detached, supplemented medium was added to stop the enzymatic reaction and the cell suspension was centrifuged for 5 min at 1500 rpm. The cell pellets were resuspended in the appropriate volume to perform seeding in the microfluidic devices.