Lps from e coli 055 b5

Unless stated otherwise, all mice used were female C57BL/6J strain (Harlan, UK), brought into the animal facility at 6 weeks of age and maintained on the open shelf with food and water ad libitum a week prior to procedure. Breeding pairs of C57BL/6 Ccr2^−/− (B6.129S4-Ccr2tm1Ifc/J) mice were a gift from Derek Gilroy, UCL and C57BL/6 Ccl2^−/− mice from Kath Else, University of Manchester. Knockout mouse lines were bred and kept homozygous for the relevant chemokine loci, and experiments performed on mice aged between 2 and 6 months of age. Mouse strains were tested to be free of the Crb1RD^8/RD8 mutation (Luhmann et al., 2012 (link)). All procedures conformed to the Association for Research in Ophthalmology (ARVO) statement for use of animals in ophthalmic and vision research.
Under anaesthesia, a 34-gauge needle (Hamilton, Switzerland) was passed perpendicular to the sclera, 0.6 mm from the corneal limbus into the vitreous cavity. 2 μl PBS containing 1 ng LPS from E.coli 055:B5 (Sigma-Aldrich, UK) or recombinant human IL-10 (Invitrogen Life Technologies, UK) was injected.

For vaccination, we used the HELP-E7SH and E7SH DNA vaccines^{21 (link),22 (link)}. OVA DNA vaccines were generated by the C-terminal addition of the OVA_257-264 coding sequence to the HELP-E7SH and E7SH DNA constructs. For DNA “tattoo” vaccination, the hair on a hind leg was removed using depilating cream (Veet) on day −1. On days 0, 3, and 6, mice were anesthetized, and 15 μl of a 2 mg/ml DNA solution in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, was applied to the hairless skin with a Permanent Make Up tattoo machine (Amiea, MT Derm GmbH), using a sterile disposable nine-needle bar with a needle depth of 1 mm and oscillating at a frequency of 100 Hz for 45 s. For rechallenge (at day 50 after primary vaccination), the hair removal step was repeated, and mice received a single DNA tattoo and an i.p. injection with 5 μg LPS from E. coli 055:B5 (Sigma) in 100 μl Hank’s Buffered Salt Solution (HBSS) on the next day (day 0, rechallenge).

The biological activity of recombinant proteins were assessed by measuring the accumulation of nitrite NO2, a stable nitric oxide (NO) metabolite produced by RAW macrophages as described previously [27] . Briefly, cells (1×10⁵cells/200 µl/well) were seeded in 96-well plates and grown overnight at 37°C/5%CO2. Then cells were incubated with either medium alone or recombinant proteins at different concentrations (10, 25, 50, 100 and 200 ng/ml) together with 100 ng/ml lipopolysaccharides (LPS from E. coli 055:B5, Sigma). After 24 h, the secreted nitrite was measured using Griess reagent (1% sulfanilamide; 0.1% naphthylethylendiamine dihydrochloride; 3% H3PO4). The absorbance was determined at 550 nm using an ELISA plate reader.

Drosophila S2 cells were maintained at 28°C in Schneider’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen). For immune stimulation, cells were incubated with 10μg/ml commercial LPS from E. coli 055:B5 (Sigma, St. Louis, MO), which is characteristic components of the cell wall of Gram-negative bacteria, for 6 h [76 (link), 77 (link)].