Anales RANF

Effect of the β -amyloid peptide on microglia activation: ATP release @Real Academia Nacional de Farmacia. Spain 191 purification, the total RNA was quantified and 1 µg was reversed transcribed using M-MLV reverse transcriptase, 6 µg of random primers and 350 µM deoxynucleotide triphosphates (dNTPs) (all from Thermo Fisher Scientific). Quantitative real-time PCR (qPCR) reactions were performed in final volume of 25 µl using the LuminoCt qPCR Ready Mix (Sigma–Aldrich), 5 µl of the cDNA synthesized previously and 1.25 µl of specific commercial TaqMan ® gene expression assays for mouse: VNUT (Mm00805914_m1), P2X7 (Mm00440582_m1), P2Y 2 (Mm02619978_s1) and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Mm99999915_g1), all from Applied Biosystems. The qPCR assays were carried out using a StepOnePlus Real- Time PCR System (Applied Biosystems) as follows: denaturation at 95°C for 20 s, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The expression of each gene was normalized to that of the endogenous housekeeping gene GAPDH amplified in parallel. 2.3. Western Blotting Microglial cells were lysed and homogenized for 1 h at 4°C in lysis buffer (pH 7.4) containing 50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P40 and Complete TM Protease Inhibitor Cocktail Tablets (Roche Diagnostics GmbH). The cell lysate was then centrifuged at 16,000 × g for 9 min and the supernatants collected. Protein extracts were resolved on 10% SDS-PAGE gels and transferred to nitrocellulose membranes, which were then blocked for 1h at room temperature ( ∼ 25°C) with 3% bovine serum albumin (BSA) in Tris buffered saline (TBS) (10 mM Tris, 137 mM NaCl, 2.7 mM KCl, 5 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 [pH 7.5]) with 0.1% Tween 20. The membranes were probed with primary antibodies raised against VNUT (1:500, MBL) or P2X7 (1:1000, Alomone Labs). Antibody binding was detected by ECL chemiluminescence (Amersham GE Healthcare), and images were captured with ImageQuant LAS 500 (GE Healthcare Life Sciences) and analyzed using ImageQuant TL. 2.4. Immunofluorescence Microglial cells cultured on coverslips in 35 mm dishes were fixed for 15 min with 4% paraformaldehyde and then rinsed with phosphate buffered saline (PBS) twice for 10 min. Afterward, coverslips were blocked and permeabilized for 1h at room temperature with blocking solution (0.1% Triton X-100, 5% fetal bovine serum and 10% bovine serum albumin in PBS). The coverslips were then incubated overnight at 4°C with the corresponding primary antibodies: rabbit anti-VNUT (1:100, MBL), mouse anti-Iba1 (1:300, Wako) or rat anti-CD11b (1:500, ThermoFisher Scientific). The following day, the cells were washed three times with PBS and incubated with the corresponding fluorescent-tagged secondary antibodies (Thermo Fisher Scientific) for 1h at room temperature. The nuclei were counterstained with 4 ′ , 6-diamidino-2- phenylindole (DAPI, Thermo Fisher Scientific) and the coverslips were finally mounted on glass slides using FluoroSave TM Reagent (Calbiochem). Images were acquired on a Leica TCS SPE confocal microscope. 2.5. ATP release Adenosine triphosphate release was measured using the ENLITEN ® rLuciferase/Luciferin reagent (Promega). Microglial cells were maintained for 30 min at 37°C in Mg 2+ -free Locke’s buffer (140 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 5.5 mM glucose and 10 mM HEPES [pH 7.4]) containing 100 µM ARL67156, a competitive inhibitor of ecto-ATPases (32), and subsequently, 50 µl of extracellular medium was collected to measure the ATP levels. The samples were centrifuged at 600 × g for 5 min at 4°C and 10 µl of supernatant were transferred onto 96-well plate placed on ice. The plate was then situated in a FLUOstar OPTIMA Microplate Luminometer (BMG LABTECH GmbH) and 100 µl of rLuciferase/Luciferin reagent was automatically injected into each well at room temperature. 2.6. Statistical Analysis The data shown are mean values ± standard error of the mean (s.e.m) and all independent experiments shown were reproduced 3–6 times. The figures were generated and the statistical analyses performed using GraphPad Prism 6 (GraphPad Software). The results were analyzed using an unpaired Student’s t -tests. A p -value ≤ 0.05 was considered statistically significant. 3. RESULTS 3.1. Effect of LPS treatment in the expression of purinergic receptors and VNUT in the microglia Microglial cells were stimulated with 1µg/ml LPS for 24h and mRNA levels of the P2X7 receptor and VNUT were analyzed. In order to confirm the effectiveness of the treatment, mRNA levels of the purinergic P2Y 2 receptor, which expression increases after stimulation with the lipopolysaccharide (33), were also measured. As can be observed in Figure 1, LPS treatment induced a significant increase in metabotropic P2Y 2 receptor expression, whereas P2X7 mRNA levels were not affected. On the other hand, VNUT mRNA levels were significantly reduced after LPS stimulation (Figure 1A). The protein levels of the nucleotide vesicular transporter and the P2X7 receptor were also measured by Western blot analyses and a marked reduction in the amount of both proteins was observed after LPS treatment (Figures 1D-F).