Saponin

CTAB (CAS: 57-09-0), was purchased from Acros Organics (Portugal), while cinnamaldehyde (CAS: 14371-10-9) and bovine serum albumin (BSA) were purchased from Sigma Aldrich (Portugal). EDTA was acquired from Panreac. Lecithin, polysorbate 80, thiosulphate, saponin, isopropanol, and DMSO were obtained from VWR Chemicals. L-histidine was purchased from Merck. All reagents were of analytical grade. Solutions of CTAB, BSA, and EDTA were prepared in sterile deionized water and cinnamaldehyde in DMSO or isopropanol. The biocide and phytochemical neutralization step was performed using the universal neutralizer (Lecithin 3 g L⁻¹, polysorbate 80 30 g L⁻¹, thiosulphate 5 g L⁻¹, L-histidine 1 g L⁻¹, and saponin 30 g L⁻¹ in 1% phosphate buffer 0.25 M, pH 7.2) for 10 min, which was shown to be efficient in inactivating the biocidal activity and to be nontoxic to the test bacteria (data not shown).

Needle-free connectors were immersed into bijous containing 1 mL of neutralizing solution consisting of 30 g/L Tween 80, 30 g/L saponin, 3 g/L lecithin, 1 g/L L-histidine, 5 g/L sodium thiosulphate in tryptone sodium chloride (all VWR International). Nullification of antimicrobial activity and non-microbial toxicity was verified prior to commencement of the study (unpublished data). The bijous were then sonicated for 10 min at 50 Hz. The entire volume of neutralizing solution was inoculated (in addition to dilutions from positive control connectors) onto chromogenic S. aureus plates (ChromID S. aureus [Biomerieux]) in duplicate.

Human fiber bundles were teased into individual fibers and transferred to wells in a 24-well plate containing PBS using fine forceps. FDB fibers were similarly incubated in PBS. Fibers were washed 3 × 10 min in PBS and incubated in blocking buffer 1% bovine serum albumin (Merck), 5% goat serum (16210-064, Gibco), 0.1% Na Azide (247-852, Merck), 0.04% Saponin (27534-187, VWR) for 1 hr. The muscle fibers were then incubated in blocking buffer containing primary antibodies overnight at 4°. The next day, the fibers were washed 3 × 10 min in PBS containing 0.04% Saponin and incubated in blocking buffer with Alexa 488 anti-rabbit or Alexa 568 anti-rabbit or anti mouse (Invitrogen) for 2 hr. Finally, the fibers were washed 3 × 10 min in PBS and mounted on glass slides in Vectashield (H-1000, Vector Laboratories) or imaged directly from the glass bottom dish. The following antibodies were used, raised in rabbit: GLUT4 (PA5-23052, Invitrogen), detyrosinated α-tubulin (AB48389, Abcam), Syntaxin6 (110 062, Synaptic Systems), or in mouse: GLUT4 (MAB8654, R&D Systems), α-tubulin (T9026, Merck).