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  • Primary cultures of rat astrocytes Astrocytes were prepared

    2020-08-06

    Primary cultures of rat astrocytes. Astrocytes were prepared from cerebral cortices of Sprague–Dawley rats born within 24h (Laboratory Animal Center of Zhejiang Academy of Medical Science, China) according to the methods described previously [29] with modifications. Briefly, cortical cells were trypsinized and plated onto flasks containing growth medium (high-glucose DMEM supplemented with 10% fetal bovine serum). After incubation for 12–14 days, the flasks were agitated at 260rpm for 24h at 37°C, and the adherent cells were trypsinized, and seeded in the growth medium. More than 95% of the cells were astrocytes as confirmed by immunocytochemical examinations with an anti-glial fibrillary acidic protein (GFAP) antibody. LTD4 (10−9–10−7M) was added into the culture medium in the presence or absence of pranlukast or Bay u9773 (Sigma–Aldrich Chemicals). After 24-h exposure, the AQP4 expression in astrocytes was determined. Determination of AQP4 expression. To determine AQP4 expression by immunostaining, NB-598 Maleate synthesis sections or the astrocytes cultured on coverslips (fixed by cold methanol at −10°C for 5min) were sequentially incubated with goat serum (1:20) for 2h, a rabbit anti-AQP4 affinity-purified polyclonal antibody (1:150; Chemicon International, Temecula, CA, USA) at 4°C overnight, and TRITC-labeled goat–anti-rabbit IgG (1:200; Chemicon) at room temperature for 2h. Finally, the brain sections and the astrocytes were examined under a fluorescent microscope (Nikon Eclipse TE2000-E, Japan). AQP4 protein expression was determined by Western blot analysis. Briefly, protein samples (50μg) from brains or astrocytes were separated by 12% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were reacted with a rabbit polyclonal antibody against AQP4 (1:500) and a mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, Kangchen Biotechnology Inc., Shanghai, China) at 4°C overnight. Following repeated washes, the membranes were sequentially reacted with a horseradish peroxidase-conjugated goat–anti-rabbit antibody (1:200; Zhongshan, Shanghai, China), at room temperature for 2h, and the ECL reagents; finally exposed on an X-ray film to show the AQP4 band (34.8kDa). After being thoroughly washed, the membranes were then reacted with another secondary antibody, a horseradish peroxidase-conjugated goat–anti-mouse IgG (1:2000; Chemicon), to show the GAPDH band (36kDa). The optical densities were quantitatively analyzed with a laser densitometer (UltroScan XL, Pharmacia LKB Co., Sweden). The results of AQP4 expression are reported as the percentage changes over GAPDH. Reverse transcription-polymerase chain reaction (RT-PCR). To determine the mRNA expressions of CysLT1 and CysLT2 receptors, total RNA was extracted from astrocytes or a rat brain using Trizol reagents (Invitrogen, USA) according to the manufacturer’s protocol. The cDNA synthesis and PCRs were performed as reported [30]. The primer sequences were as the following: rat CysLT1 receptor forward 5′-(+) TCT CCG TTG TGG GTT TCT-3′ and reverse 5′-(+) TAT AAG GCA TAG GTG GTG-3′ (product size 214bp); rat CysLT2 receptor forward 5′-(+) AGC GTT AGG AGT GCC TGG AT-3′ and reverse 5′-(+) CAA GTG GAT GGT CCG AAG TG-3′ (product size 520bp); β-actin forward 5′-(+) TAC AAC CTC CTT GCA GCT CC-3′ and reverse 5′-(+) GGA TCT TCA TGA GGT AGT CAG TC-3′ (product size 620bp), or forward 5′-(+) AAC CCT AAG GCC AAC CGT GAA-3′, and reverse 5′-(+) TCA TGA GGT AGT CTG TCA GGT C-3′ (product size 285bp).