Amikacin sulphate

The following antibodies were obtained from commercial sources: monoclonal mouse anti-CD59 (MEM-43, Abcam, Cat. No. ab9182), monoclonal rabbit anti-flotillin-1 (D2V7J) XP (Cell Signaling, Cat. No. 18634), monoclonal mouse anti-flotillin-2 (BD Biosciences, Cat. No. 610383), polyclonal rabbit anti-GAPDH (Sigma, Cat. No. G9545), monoclonal rabbit anti-phospho-Src Family (Tyr⁴¹⁶, Cell Signaling, Cat. No. 6943), monoclonal rabbit anti-Src Family (Cell Signaling, Cat. No. 2109), monoclonal rabbit anti-caveolin-1 (D46G3) XP (Cell Signaling, Cat. No. 3267). LecA was detected by a custom-made polyclonal rabbit anti-LecA antibody (Eurogentec, France).
The protease inhibitors Aprotinin, Leupeptin, Pefablock, Sodium orthovanadate and Phosphatase Inhibitor Cocktail 3 were all obtained from Sigma-Aldrich. RPMI 1640, DPBS-/-, FCS and l-Glutamine were all purchased from Gibco (Thermo Fisher Scientific). The following chemicals were obtained from Roth: BSA, DABCO, DAPI, EDTA, glycerol, LB, Mowiol, NaCl, sodium deoxycholate, NH₄Cl, paraformaldehyde, SDS, Tris (hydroxymethyl)-aminoethane, Triton X-100 and Tween 20. Amikacin sulphate and Gentamicin were obtained from Sigma-Aldrich. StxB was purchased from Sigma-Aldrich (Cat. No. SML0562).

P. aeruginosa strain PAO1, characterized first in 1955 [50 (link)], was cultivated, GFP-tagged and deleted of LecA as described before [8 (link)]. For the invasion assay, overnight cultures of PAO1 wild-type (WT) and ΔLecA were centrifuged before resuspension of the pellet in RPMI containing 1 mM CaCl₂ and MgCl₂. H1299 cells (with control siRNA (Qiagen), CD59- or flotillin-depleted) were treated with PA at a multiplicity of infection (MOI) of 100 for 2 h at 37 °C. After washing with PBS, cells were treated with Amikacin sulphate (400 μg/ml; Sigma-Aldrich) for 2 h at 37 °C to exclude extracellular bacteria. Finally, the cells were lysed with 0.25% (vol/vol) Triton X-100, plated on LB-Agar plates containing 60 μg/ml Gentamicin and incubated at 37 °C overnight. The following day, colonies were counted and invasion rate was calculated as percentage of Amikacin-survived bacteria to the total number of bacteria not treated with Amikacin. Mean values of three individual experiments were normalized to the invasion rate of WT PAO1 into WT H1299.

Four aminoglycosides (Gentamicin, Tobramycin, Amikacin and Apramycin) were used in this study. Gentamicin and tobramycin ampules, as well as amikacin sulphate and apramycin sulphate were obtained from Sigma-Aldrich (the Netherlands). In Pakistan, gentamicin sulphate and amikacin sulphate ampules were purchased from Ray Pharma and tobramycin sulphate ampules were purchased from AGP pharma.