Next, 20 L of MTT (5 mg/mL) was added to each well and incubated for 4 h

Next, 20 L of MTT (5 mg/mL) was added to each well and incubated for 4 h. and 180 rounds per minute. Animals and experimental design Sixty adult female postpartum and lactating BALB/c mice (6C8 weeks old, weighing 35C40 g) were obtained from Wuhan Institute of Biological Products Co Ltd (Wuhan, China). All procedures were performed according to the Guide for the Care and Use of Laboratory Animals established by the US National Institutes of Health and approved by the Ethical Committee on Animal Research at Huazhong Agricultural University. The mice were housed in a room maintained at 241 C Amlodipine besylate (Norvasc) with 40%C80% humidity. All animals received food and water cultures were maintained in 100 mL of LB broth at 37 C and 180 rounds per minute until they reached the stationary phase. Subsequently, the bacteria were centrifuged and then washed twice with phosphate-buffered saline (PBS, pH 7.0). The collected cell pellet was resuspended in 1 mL of PBS at 3.11010 CFU/mL. The method for establishing the suspension (109 CFU) was injected into two abdominal mammary glands to induce mastitis. Then, 24 h after infection, three intraperitoneal injections of PD or DEX were given every 8 h at doses established in a previous study20. The mice were randomly divided into four Amlodipine besylate (Norvasc) groups as follows. 1. Control group (CG): The mice were treated with 100 L of PBS as a vehicle control. 2. group (SG): The mouse model of mastitis without drug intervention. 3. PD groups: 24 h after infection, the mouse model of mastitis was Amlodipine besylate (Norvasc) intraperitoneally administered PD at 15, 30 and 45 mg/kg. 4. Dexamethasone administration group (DEX group): 24 h after infection, the mouse model of mastitis was intraperitoneally administered DEX at 5 mg/kg. Then, 8 h after the last treatment with PD or DEX, all the mice were euthanized with sodium pentobarbital, and the mammary gland tissues were collected and stored at -80 C for further research. Cell culture and treatment Primary mouse mammary epithelial cells were prepared as previously described21. Mammary gland tissues were aseptically harvested from normal pregnant BALB/c mice and minced into paste. The homogenized tissues were digested with a collagenase I/II/trypsin mixture at 37 C and then centrifuged. The fatty layer and cell pellets were resuspended in DMEM/F12 with 10% FBS. The suspension was centrifuged again and filtered through a 40 m pore-size filter, Rabbit Polyclonal to FA7 (L chain, Cleaved-Arg212) and the cells were then cultured at 37 C with 5% CO2. The primary mouse mammary epithelial cells were identified using specific antibodies against cytokeratin 18. Mouse mammary epithelial cells (mMECs) were pretreated for 1 h with PD at final concentrations of 25, 50 and 100 g/mL. PD-untreated cells containing DEX (100 g/mL) were used as a positive control. Subsequently, at a multiplicity of infection (MOI) of 10 was added to the cells. After 8 h of incubation at 37 C, the cell supernatant and cells were collected for further study. Cell viability assay The effect of PD on mMEC viability was evaluated with an MTT assay. mMECs were incubated in the presence or absence of various concentrations of PD (25, 50 and 100 g/mL) and DEX (100 g/mL) for 24 h. Next, 20 L of MTT (5 mg/mL) was added to each well and Amlodipine besylate (Norvasc) incubated for 4 h. After the supernatants were removed and the formazan was dissolved with 150 L of DMSO in each well, the optical density (value of each well was read at 450 nm with an automatic microplate reader (Thermo Scientific Multiskan MK3, USA). TransAM NF-B and AP-1 activation assays A TransAM NF-B p65 kit and a TransAM AP-1 Family kit were used to determine the concentrations of NF-B p65 and AP-1 in nuclear protein extracts obtained from mMECs via a colorimetric DNA-binding ELISA, which was performed according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) assays Total RNA was extracted from the mammary gland tissues and mMECs using TRIzol reagent (Invitrogen, USA), according to the manufacturer’s instructions. The concentration and purity of the extracted RNA were evaluated with Q5000 (Quawell Technology, USA). Total RNA (1 g) was reverse-transcribed into first-strand DNA (cDNA) using a Prime-Script RT-PCR kit according to the manufacturer’s instructions (Takara)..