Lysis buffer

Levels of various cytokines/chemokines in the sera and sciatic nerves tissue lysates were analyzed using a mouse antibody array glass chip (RayBio Mouse Cytokine Antibody Array G series; RayBiotech Inc., Norcross, GA, USA) in WT mice in all experimental conditions. To obtain serum sample, blood was collected via beheading immediately following euthanasia, allowed to clot at room temperature for 30 min and then centrifuged at 3000 rpm for 15 min. Lysis buffer (Raybiotech, Inc) containing proteinase inhibitor (Sigma Aldrich) was added to sciatic nerve homogenates and 50 μg of each sample was added to the array. Incubation and washes were performed following the manufacturer’s instructions described in supplemental material. Fluorescence detection was performed using an Agilent G2564B microarray scanner (Agilent Technologies Italy) and analysis was performed using the array testing services from RayBiotech.

After treatment, cells were harvested with lysis buffer (RayBiotech, GA, USA, catalog no. EL-lysis), and protein/enzyme concentrations or activities were measured in cell lysates using the following commercially available kits: HIF-1α, c-Myc, and G6PD (Abcam, Cambridge, UK, catalog no. ab171577, ab277452, and ab102529, respectively), LDH-A and PKM2 (Elabscience, Wuhan, China, catalog no. E-EL-H0556 and E-EL-H6063, respectively), and glutaminase (Cohesion Biosciences, London, UK, catalog no. CAK1065) according to the manufacturer’s instructions. Protein concentrations were quantified using a BCA Protein assay kit (Abcam, Cambridge, UK, catalog no. ab102536).

Freshly isolated mononuclear cells were pelleted at 800 g for 10 min and washed 3 times with sterile PBS. Cells were lysed in a lysis buffer (RayBiotech, Peachtree Corners, GA, United States, Cat. No. AA-LYS-10mL) supplemented with 1x Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, United States, Cat. No. P8340), 1× Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich, Cat. No. P5726), 1× Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich, Cat. No. P0044), and incubated in ice for 30 min. The lysates were centrifuged at 15,000× g at 4 °C for 30 min and stored at −80 °C. Cell lysates were sent to the tebu-bio facility in order to perform Multiplex ELISA array and subsequent technical normalizations.

Murine hearts were homogenized in lysis buffer (Ray Biotech, USA), and total proteins were extracted, according to the manufacturer's instructions. The levels of TNF-α, IL-1β, and MMP-9 were assayed using commercial ELISA kits, according to the manufacturer's instructions (Ray Biotech, USA).

Eyes were enucleated and washed two times with ice-cold PBS. Samples were mechanically lysed in lysis buffer (RayBiotech, Norcross, GA, USA) containing a protease inhibitor cocktail (Thermo, Waltham, MA, USA) and centrifuged at 12,000 g for 10 minutes. The levels of NGF, bFGF, GDNF and TSP-1 were measured in the supernatant of the lysates using the Mouse beta-NGF ELISA kit (RayBiotech), Mouse FGF basic Quantikine ELISA kit (R&D Systems, Minneapolis MN, USA), Mouse GDNF ELISA kit (MyBioSource, San Diego, CA, USA) and Mouse thrombospondin 1 ELISA kit (MyBioSource), respectively. Data were normalized per mg of protein present in each sample.

Lipid peroxidation was determined by a method that measures the amount of thiobarbituric acid reactivity by the amount of malondialdehyde (MDA) formed during acid hydrolysis of the lipid peroxide compounds using the Lipid Peroxidation MDA Assay kit (Cayman, Ann Arbor, MI, USA). Retinas were mechanically lysed in lysis buffer (RayBiotech) containing a protease inhibitor cocktail and the antioxidant butyl hydroxytoluene (BHT). Lipid peroxidation was quantified sprectophotometrically at 540 nm (Thermo spectronic). MDA level was normalized per mg of protein present in each sample.
Protein nytrosilation was quantified in retinal lysates containing a protease inhibitor cocktail, measuring nitrotyroxine level with the 3-Nitrotyrosine ELISA kit (Abcam, Cambridge, UK) following the manufacturer’s instructions. The nitrotyroxine level was normalized per mg of protein present in each sample.
DNA oxidation was measured in genomic DNA isolated from retinal samples by DNAzol (Invitrogen). DNA was digested by incubation with DNAsa RQ1 (Promega, Madison, WI, USA) and 8-OHdG levels were quantified using the Oxidative DNA damage ELISA kit, following the manufacturer’s instructions. 8-OHdG levels were normalized per μg of DNA present in each sample.
For all oxidative markers, data were expressed as fold-change versus normal mice.