Fm4 64fx

All staining was performed in 96-well plates with 50 µL of fixed Mtb cells diluted in 50 µL of PBST. Staining was performed with 0.6 μg of FM4-64FX (ThermoFisher; F34653) and 15 μL of a 0.1 μM SYTO 24 (ThermoFisher; S7559) stock in each well containing PBST and fixed bacilli. The plate was then incubated at room temperature in the dark for 30 min. Once stained, the cells were washed once with an equal volume of PBST and resuspended in 30 µL of PBST. Stained Mtb were spotted onto agar pads (1% wt/vol agarose; SigmaAldrich; A3643-25G). Images were captured with a widefield DeltaVision PersonalDV (Applied Precisions) microscope. Bacteria were illuminated using an InsightSSI Solid State Illumination system with transmitted light for phase contrast microscopy. SYTO 24 was imaged using Ex. 475 nm and Em. 525 nm. FM4-64FX was imaged with Ex. 475 nm and Em. 679 nm. Montage images were generated using a custom macro that captures 25 individual fields of view per image. Two technical replicate images were taken from each sample for a total of 50 images per biological replicate. Three biological replicates were generated for each drug treatment. Images were recorded with a DV Elite CMOS camera for all three channels.

The hearts from AAV-infected mice were isolated and perfused with FM4-64FX and subjected to in situ confocal microscopy. The t-tubule content, integrity and regularity were next analyzed in ImageJ following previously described STAR Methods.^{79 (link)} Briefly, intact mouse hearts were Langendorff-perfused at room temperature with Tyrode’s solution (NaCl 137, KCl 5.4, HEPES 10, Glucose 10, MgCl₂ 1, NaH₂PO₄ 0.33, pH adjusted to 7.4 with NaOH, oxygenated with 95% O₂ and 5% CO₂ during experiments), containing 10 μM FM4-64FX, a lipophilic fluorescence indicator of membrane structure (Thermo Fisher), for 20 min. The membrane structure of epicardial myocytes was analyzed in situ with confocal microscope (STELLARIS 8, Leica Microsystems). T-tubule images were next analyzed with IDL image analysis program (ITT VIS Inc., Colorado). Background noise in confocal images was filtered with a threshold value retrieved from image intensity histogram. T-tubule two-dimensional images were converted to frequency domain using the Fast Fourier Transformation function in IDL, so that it could be determined whether repeating patterns occur (T-tubule regularity) and how strong the repeating patterns are (T-tubule power).

Animals were mounted in 3% low melting point agarose in FNSW (2-Hydroxyethylagarose, Sigma Aldrich A9414) between a slide and a cover slip (using five layers of adhesive tape for spacing) and imaged with a Leica SP8 confocal microscope. Fluorescent labeling of musculature was achieved either by microinjection of mRNAs encoding GCaMP6s, LifeAct-EGFP or H2B-RFP, or by incubation in 3 μM 0.1% FM-464FX (ThermoFisher Scientific, F34653). Contraction speed was calculated as (l2-l1)/(l1*t), where l1 is the initial length, l2 the length after contraction, and t the duration of the contraction. Kymographs and wave speed quantifications were performed with the ImageJ Kymograph plugin: http://www.embl.de/eamnet/html/kymograph.html. No sample size was computed before the experiments. At least two technical replicates were performed for each assay, with at least two biological replicates per sample per technical replicate. A technical replicate is a batch of treated individuals (together with their control siblings), and a biological replicate is a treated (or control sibling) individual.

As described [17 (link)], cultured neurons (DIV14–15) expressing Syn1-WT-GFP or Syn1-3CA-GFP were stimulated for 1 min with 55 mM KCl, in the presence of 10 μM FM4-64 FX (Thermo Fisher Scientific, Cat # F34653) and 1 μM TTX (Cayman, Cat # 14964) in Krebs’–Ringer’s–HEPES solution (KRH, 130 mM NaCl, 5 mM KCl, 1.2 mM MgSO_4, 1.2 mM KH₂PO₄, 2 mM CaCl₂, 6 mM glucose, and 25 mM HEPES, pH 7.4). Followed by two times complete medium substitutions, neurons were perfused for 10 min with warmed KRH (37 °C) supplemented with 1 μM TTX and 10 μM CNQX (MCE, Cat # HY-15066) and fixed with 4% paraformaldehyde for 10 min on ice. After 3 times washing with ice-cold HBSS, the coverslip was mounted for imaging. FM4-64 fluorescence was measured on digital images within an area of 3 × 3 pixels at the center of synapses expressing Syn1-WT-GFP or Syn1-3CA-GFP as indicated.

To assess downstream targets of ROS, membrane disruption, and DNA damage, in C. difficile 2015, bacterial cells were washed thoroughly in PBS and adjusted to OD_600 nm of 1 in sterile anaerobic PBS and treated with 10 mM reuterin or 10 mM reuterin and 20 µM glutathione. As a positive control, C. difficile was also incubated with 500 μM H₂0₂. All cultures were incubated for 1 hr at 37ºC anaerobically. For membrane staining, C. difficile was incubated with 5 µg/ml FM 4–64FX (ThermoFisher #F34653) for 1 min on ice. Treated cells were then centrifuged at 5,000 x g for 5 min and fixed in 4% paraformaldehyde (PFA) at room temperature for 1 hr. Bacterial cells were counter-stained with Hoechst 33342 (Invitrogen) for 10 min at RT. Fixed bacteria were mounted on slides with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich # F4680) and imaged on a Nikon Eclipse 90i. For DNA damage, C. difficile was incubated with 4% PFA at room temperature for 1 hr and dried on slides. Cells were permeabilized, treated with TUNEL staining reagents as previously described^{81 (link)}, and imaged on a Nikon Eclipse 90i. TUNEL stained images were quantified using FIJI (formerly known as ImageJ, National Institutes of Health) software by tabulating the mean pixel intensity in five regions per slide (n = 2 slides/experiment; performed two independent times).

FM4-64FX (Thermo) was diluted to 5 μg/mL in Hank’s balanced salt solution in the dark. HK-2 cells were washed with Hank’s solution and 1 mL of FM4-64FX was added to each dish. Laser confocal microscopy (Olympus, Tokyo, Japan) was used to observe endocytosis. The results were analyzed by Image-Pro-Plus software (Version 6.0, Media Cybernetics, Rockville, MD, USA).