Anaerocult a mini system

A normal rat renal proximal tubule cell line NRK-52E was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Cells were cultured in DMEM supplemented with 5% fetal bovine serum and antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin) at 37 °C in 5% CO₂. In some experiments, the cell culture in a hypoxia/reoxygenation (H/R) condition was performed. The oxygen content was reduced to 0.2% to provide hypoxic condition using an Anaerocult A mini system (Merck, Whitehouse Station, NJ). After 6 h of hypoxia, cells were removed from the hypoxic condition followed by a medium change for 24 h reoxygenation.

For immunoblotting assays, Giardia trophozoites were plated in sterile 6-well plates at a density of 1×10⁶ cells mL⁻¹ in a volume of 3 mL medium/well. Incubation was performed at 37°C allowing the plates to equilibrate for 24 h either under anaerobic conditions (Anaerocult A minisystem, Merck) or with air (atmospheric O₂ level), in the presence or absence of 120 U mL⁻¹ catalase.
After incubation, trophozoites were detached on ice for 30 minutes, collected by centrifugation and lysed (lysis buffer C3228, Sigma). After total protein content determination by the bicinchoninic acid assay, cell extracts (20 µg protein/lane) were subjected to SDS–PAGE and proteins blotted onto a polyvinylidene difluoride (PVDF) membrane (Immobilon pSQ, Merck). Blots were then incubated with rabbit polyclonal antibodies raised against GiPrx1a or deltaGiPrx1b (Davids Biotechnologie GmbH), followed by incubation with alkaline peroxidase-conjugated secondary antibodies (NA934, GE Healthcare) and detection by enhanced chemiluminescence (ECL kit RPN2132, GE Healthcare). In these assays, using either the anti-GiPrx1a or the anti-deltaGiPrx1b antibodies was irrelevant, because in dot-blot experiments each of the two antibodies showed cross-reactivity with both GiPrxs.

The anti P. acnes activity of the gel formulations containing the herbal ball extract was evaluated by using the agar well diffusion method. The BHI agar was inoculated by spreading P. acnes over the entire agar surface and left to dry. A hole with a diameter of 6 mm was punched aseptically with a sterile tip. The gel formulations were introduced into the well. Then, agar plates were incubated in an anaerobic environment using the Anaerocult A Mini system (Merck, Darmstadt, Germany) at 37 °C for 48 h. Gel base and Clindalin^® gel (clindamycin 1% gel) were used as the placebo and positive control, respectively. The anti P. acnes activity was investigated as the diameters of the clear zone.