Concanavalin a (cona)

S2 cells were maintained in Schneider’s medium (ThermoFisher, 21720-001) supplemented with 10% (v/v) fetal bovine serum (ThermoFisher, 16140071) and 1% (v/v) penicillin streptomycin (ThermoFisher, 15140122). Constructs expressed in S2 cells were driven by the act5c promoter by subcloning into Ac5-Stable2-neo (Addgene #32426). Transfections were carried out using Effectene (Qiagen, 301425) following manufacturers recommendations. Transfected S2 cells were plated onto Labtek slides (ThermoFisher, 155409) coated with a 50 μg/ml solution of Concanavalin A diluted in PBS (Cayman Chemical, 14951) for 1 h at room temperature. S2 cells were subsequently imaged on a widefield Nikon Eclipse Ti using a Plan-Apochromat 60 × /1.4 NA oil objective.

In order to visualize potential survival of S. maltophilia in amoebae, confocal microscopy was performed on co-cultures. After the incubation periods (0 to 96 hours), cells within the 24-well microplates were fixed with 4% paraformaldehyde (Electron Microscopy sciences, Hatfield) for 30 minutes. Cells were then washed twice with Phosphate Buffer Saline (PBS: 8 g.L^-1 NaCl, 0.2 g.L^-1 KCl, 1.44g NA₂HPO₄, 0.24 g.L^-1 KH₂PO₄) and were permeabilized for 10 minutes with 0.1% Triton. Coverslips were incubated for one hour at room temperature in PBS in wet room with primary rat antisera directed against total proteins of S. maltophilia (Abcam, Cambridge, United Kingdom). After washing twice with PBS, coverslips were incubated in wet room for one hour with second anti-rat antibodies coupled to Alexa Fluor 488 (488- emission 505) (Abcam, Cambridge, United Kingdom) in PBS containing concanavalin A (Cayman Chemical company, Ann Arbor, Michigan) in order to label amoebae red. After three washings with PBS and one with deionized water, the coverslips were mounted onto glass slides using the mounting medium Mowiol (Sigma-Aldrich, MO). The observations were performed on a Zeiss confocal microscope LSM800 (Munich, Germany) using a x63 apochromatic objective (NA 1.4), 0.7 μm optical sections and photos were analyzed using Zen software for microscopy. All experiments were performed in triplicate.