aureus 8325-4 and DU 1090 were cultured in TSB at 37 °C to an opt

aureus 8325-4 and DU 1090 were cultured in TSB at 37 °C to an optical density at 600 nm of 0.5. Fifty-milliliter culture aliquots were centrifuged, NVP-BKM120 datasheet washed with PBS, and resuspended in 1 mL PBS (2 × 108 CFU per 30 μL)

for histopathology experiments. For cytotoxicity studies, 5 mL of culture described above was resuspended in 10 mL of Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, CA). The minimal inhibitory concentrations (MICs) of apigenin for S. aureus were evaluated using the broth microdilution method according to CLSI guidelines (CLSI, 2005). Briefly, apigenin was diluted in a 96-well plate over the concentration range of 4-1024 μg mL−1 using double dilution method. Following inoculation with 5 × 105 CFU mL−1 of overnight broth cultures in each well, the plate was inoculated at 37 °C for 24 h. The MIC was defined as the lowest concentration at which the growth of S. aureus was inhibited. Staphylococcus aureus strain 8325-4 was cultured in TSB medium at 37 °C, shaken at 200 r.p.m. to an optical density (OD600 nm) of 0.3, and aliquoted into five 250-mL flasks in a volume of 100 mL. Apigenin dissolved in DMSO was added to the four cultures to obtain final concentrations of 1, 4, 16, and 64 μg mL−1. 1% DMSO was added to the control culture. The bacteria were cultured at 37 °C with constant shaking, and cell growth

was measured by reading the OD600 nm values every 30 min. Hemolytic activity was measured as described previously (Worlitzsch et al., 2001; Qiu et al., 2010a) using rabbit Erastin erythrocytes. Briefly, S. aureus cultures with different concentrations of apigenin were harvested when grown to the postexponential growth phase by centrifugation

(5500 g, 4 °C, 1 min), and the residual cells were removed using a 0.2-μm filter. A 0.1 mL volume of bacterial culture supernatants were brought up to 1 mL with the addition of PBS and 25 μL defibrinated rabbit erythrocytes for 30 min at 37 °C. The unlysed blood cells were removed by centrifugation (5500 g, room temperature, selleckchem 1 min). Following centrifugation, the hemolytic activity of the supernatants was detected by measuring the optical density at 543 nm. Medicine-free culture supernatant served as the 100% hemolysis control. The percent of hemolysis was calculated by comparison with the control culture supernatant. The culture supernatants collected previously were used in Western blot analysis. Samples were boiled with Laemmli sample buffer for 10 min, and then 25 μL of the sample was fractionated by SDS-PAGE (12% polyacrylamide gels; Laemmli, 1970). The Western blot protocol was performed as described previously (Qiu et al., 2010a, b). Proteins were transferred onto polyvinylidene fluoride membranes (Roche, Basel, Switzerland) using a semi-dry transfer cell (Bio-Rad, Munich, Germany). The membrane was blocked for 2 h with 5% bovine serum albumin (Amresco) at room temperature.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>