Bis benzamide

Primary cerebellar cells were plated into 96-well poly-l-lysine–coated black plates (Costar) and cultured for one (neurons) or seven (astrocytes) days prior to overnight incubation with 100 μM d-α-tocopherol (Acros Organics). The following day, the cells were incubated in 10 μg/ml 2',7'-dichlorodihydrofluorescein diacetate in HBSS without phenol red (Invitrogen) for 1 h, washed, and challenged with the indicated concentration of H₂O₂ for 1 h. After a final wash in HBSS, DCF fluorescence was measured on a PerkinElmer Victor 3 plate reader (excitation = 485 nm; emission= 535 nm) and normalized to DNA content as measured after incubating cells with bisbenzamide (Sigma; 2.5 μg/ml in 2 M NaCl, 50 mM Na₂HPO₄ [pH 7.4] in the dark at 37 °C for 60 min) in a plate reader (excitation = 365 nm; emission = 460 nm).
Statistical analyses and graphing were done using GraphPad Prism 6 (GraphPad Software, Inc). Student's t test and one-way ANOVA with Tukey's multiple comparisons post hoc analysis were performed, and threshold for statistical significance was set at p < 0.05.

After treatments for 16 hrs, myotubes were loaded with DCFH-DA dye (Invitrogen) for 1 h to measure ROS levels. Amount of ROS was normalized to total cellular DNA which was measured using bis-benzamide (Sigma). bis-benzamide was added for 15 min. Myotubes were lysed and lysates were transferred to 96-well black well plate. DCF fluorescence was measured at 485 nm excitation and 528 nm emission. DNA levels were measured at 360 nm excitation and 460 nm emission. Data are represented as ROS fluorescence (RFU)/DNA fluorescence (RFU).

Quantification of viable Cpn was performed in an identical manner to our previous report (Little et al., 2005 (link)) in the following manner. Frozen tissue was thawed and a 10% weight to volume homogenate was prepared in serum-free MEM (Thermo Fisher Scientific, Pittsburgh, PA, USA) supplemented with 2 mM Glutamine. Serial 10-fold dilutions (in 200 μL) were added to four well Lab Tech chamber slides (Naperville, IL, USA) on which HEp-2 cells were previously plated. Negative control wells contained cells mock-infected with medium alone. Chamber slides were incubated at 37°C in 5% CO₂ for 2.5 h, washed with HBSS and refilled with fresh complete medium supplemented with 2 μg/ml cycloheximide (Sigma-Aldrich, St. Louis, MO, USA) followed by incubation for 48 h at 37°C. After incubation, slides were washed with HBSS, fixed in 50% methanol at RT for 20 min, washed twice in HBSS, and labeled with a 1:10 dilution of FITC-conjugated Chlamydia-specific antibody (Imagen^TM; DAKO, Carpenteria, CA, USA) for 90 min at 37°C, protected from light. Slides were washed in phosphate buffered saline (PBS) and counterstained with a 2 μg/ml of Bisbenzamide (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 min, washed in PBS and coverslipped with aqueous mounting medium (Imagen^TM; DAKO, Carpenteria, CA, USA). All titers are calculated as IFUs/ml of 10% weight to volume tissue homogenate.