Gentamicin

Optic nerve explant cultures were prepared from mice aged postnatal day (P)7–11, as described previously (Hawkins and Butt 2013 (link)). In brief, optic nerves were carefully dissected and maintained in pre-warmed (37 °C) and pre-gassed (95 % O₂/5 % CO₂) dissecting media, constituted of Dulbecco’s Modified Eagle Medium (DMEM) (Sigma) medium, supplemented with 4 mM l-glutamate, 10 % foetal bovine serum (FBS; Invitrogen) and 0.1 % gentamicin (Invitrogen, Life Technologies Ltd., Paisley). From this point on optic nerves were kept under sterile conditions and cut into 1–2 mm fragments in filter sterilized pre-warmed dissecting media, using a scalpel blade. For further dissociation, optic nerve fragments were triturated and nerve fragments transferred onto laminin (Invitrogen) and poly-l-ysine (Sigma) coated coverslips and incubated in dissecting medium at 37 °C in 95 % O₂/5 % CO₂ overnight to allow the adhesion of the explants. After 24 h, the dissecting media was substituted with a low serum (0.5 %) modified Bottenstein and Sato (B&S) culture medium, supplemented with 10 ng/ml recombinant human PDGF-AA (R&D Systems) and 0.1 % gentamicin. After 3–4 days in vitro (DIV) the medium was replaced to B&S media supplemented with 0.5 mM dibutyryl cAMP for up to 10 DIV. Explanted cells were then cultured in B&S media with 0.1 % gentamicin for up to 15 DIV, changing media every 3–5 days.

To determine bacterial uptake by phagocytes, primary human neutrophils, primary human monocyte-derived macrophages, or THP-1 human monocyte-like cells were infected with S. Typhi. Cells were seeded in 24-well plates at a density of 5 × 10⁵ cells per well. Bacteria were added to cells at a multiplicity of infection (MOI) of 10 bacteria per cell. The plate was centrifuged at 250 × g for 5 min and incubated (37°C, 5% CO₂) for 30 min. After this incubation, RPMI medium (0.5 mL) containing 0.1 mg/mL gentamicin (Gibco) was then added to the cells for 30 min at 37°C to kill extracellular bacteria. Cells were washed three times with 0.5 mL of PBS and then lysed with 0.5 mL of pre-chilled sterile water for 15 min. The recovery of bacteria from phagocytes was quantified by spreading serial 10-fold dilutions onto LB agar plates with the appropriate antibiotics to enumerate CFU.
For blocking antibody assays, gentamicin protection assays were performed as described above with the addition of either 5 μg of anti-human DC-SIGN/CD209 antibody (clone no. 120507; R&D Systems) or 5 μg of anti-mouse IgG2B isotype control antibody (clone no. MAB004; R&D Systems) per well for 30 min prior to infection with bacterial strains.

Fresh peripheral
blood mononuclear
cells, obtained by a ficoll gradient, from 40 mL of blood per individual,
were used for monocyte purification by means of anti-CD14 microbeads
following the manufacturer’s protocol (Miltenyi Biotec, Bergisch
Gladbach, Germany). The CD14– cell fraction was placed in 10%
DMSO and frozen for a later lymphocyte proliferation test. To generate
DCs, monocytes (CD14+ cells) were incubated in complete medium containing
Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific,
Carlslab, CA) supplemented with 10% fetal bovine serum (Thermo Fisher
Scientific), streptomycin (100 μg/mL), and gentamicin (1.25
U/mL), as well as recombinant human rhGM-CSF (200 ng/mL) and rhIL-4
(100 ng/mL) (both from R&D Systems Inc., Mineapolis, MN) for 5
days at 37 °C and 5% CO₂.