Mab479

Dorsal air pouches were prepared by injection of 2.5 mL of air on day 0 and day 3 in CD-1 mice, as previously described.25 (link) On day 6, mice received 0.25 mL of one of the following treatments diluted in 0.5% carboxymethyl cellulose (CMC, Sigma-Aldrich): (1) vehicle, CMC alone; (2) IL-17 (1 µg); (3) nIL-17 (1 µg); (4) IL-17 (1 µg) plus MAB421 or Ab-IPL-IL-17 (10 µg); (5) IL-17 (1 µg) plus anti-JE (10 µg, MAB479, R&D System) and (6) IL-17 (1 µg) plus anti-KC (10 µg, MAB453, R&D System). Mice were sacrificed after 24 hours, and lavage fluids were recovered, and centrifuged at 220 g for 10 min at 4°C. Cell pellets and inflammatory exudates were banked for subsequent analysis of inflammatory cyto-chemokines. The route, timing and frequency of administration as well as the selected dosages of tested compounds were selected according to updated literature.12 16 (link) Cell number was determined by TC20 automated cell counter (Bio-Rad) using Bio-Rad’s TC20 automated cell counter uses disposable slides, TC20 trypan blue dye (0.4% trypan blue dye w/v in 0.81% sodium chloride and 0.06% potassium phosphate dibasic solution, Sigma-Aldrich) and a CCD camera to count cells based on the analyses of capture images.25 (link)

In primary hippocampal cultures (10–11 DIV) conditioned medium was removed and stored for later usage; neurons were washed and stimulated with Glu (100 μM, 30 min) in modified Locke’s buffer (without MgCl₂ plus 1 μM glycine to stimulate all types of Glu receptors), in the presence or in the absence of recombinant mouse mCX3CL1 (100 nM, Peprotech). Under these experimental conditions, only neurons die (Chen et al., 2000 (link); Rosito et al., 2012 (link)). After treatment, cells were re-incubated in the original conditioned medium for 18–20 h, treated with lysis buffer (0.5% ethylhexadecyldimethylammonium bromide, 0.28% acetic acid, 0.5% Triton X-100, 3 mM NaC1, 2 mM MgCl, in PBS pH 7.4) and counted in a hemocytometer for viability, as described (Volontè et al., 1994 (link)). Data were expressed as percentage of viable cells taking as 100% the number of viable cells in control cultures. Variability in the number of viable cells in control conditions never exceeded 10%.When necessary, cells were pre-treated with monoclonal mouse αCCL2 Ab (3 μg/ml, 30 min; R&D MAB479), rat IgG (3 μg/ml, 30 min; Santa Cruz Biotecnology sc-2032), 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2phenyl-4-propyl-3-pyrinide carboxylate (MRS1523; 100 nM, Sigma) in culture medium; drugs were present also during and after Glu challenge.

Frozen retinas were homogenized with a multi-beads shocker (Yasui Kikai Corp.), and total RNA was extracted. mRNA expression of angiogenic factors was analyzed by real-time PCR using target-specific primers (Takara Bio).
Primary retinal microvascular endothelial cells isolated from C57BL/6 mice (Cell Biologics Inc.) were cultured in a specified endothelial cell medium supplement kit (Cell Biologics Inc.) and passaged no more than 3 times. After washing with PBS, recombinant mMCP6 was added to the culture (final concentration of 300 ng/ml) and incubated overnight. Cells were collected and processed for real-time PCR analysis. To assess effects of MCP1, 10 μg/ml rat anti-MCP1 mAbs (R&D Systems, catalog MAB479, clone 123616) were added into the culture. Rat IgG (Abcam, catalog ab18450, clone RTK2758) was used as an isotype control.