Polycarbonate membrane filters

Senicapoc was a gift from Prof. Heike Wulff, University of California, Davis. Dipalmitoylphosphatidylcholine (DPPC) and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Carboxyfluorescein) were purchased from Avanti Polar Lipids (Abalaster, AL, USA). Whatmann drain discs and polycarbonate membrane filters were purchased from GE Healthcare Life Sciences (Freiburg, Germany). Chloroform (HPLC Grade) was purchased from Tedia Chemicals (Fairfield, OH, USA). Acetonitrile and Methanol (LCMS Grades) were purchased from Fisher Scientific (Hampton, NH, USA). Cellulose ester dialysis tubings (100 kDa MWCO) and tubing closures were obtained from Spectrum Laboratories (Rancho Dominguez, CA, USA). Salts used to make Phosphate Buffered Saline (PBS) includes sodium chloride (NaCl), potassium chloride (KCl), Di-sodium hydrogen phosphate (Na₂HPO₄) and Potassium dihydrogen phosphate (KH₂PO₄) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Zinc Sulphate Monohydrate salt for analytical processing was also purchased from Sigma-Aldrich. Soft tissue homogenizing kits (CK14, 0.5 mL) used for tissue analysis were purchased from Bertin-Instruments (Rockville, MD, USA).

An aliquot of 1.5 mL was collected from each culture, fixed with 4% formaldehyde (v/v) and stored at −20°C until DAPI staining. After thawing, the filaments were harvested on polycarbonate membrane filters (0.88 μm pore size, 22.5 mm circle diameter, GE Healthcare, Chicago, IL, United States) and stained with 50 μl of DAPI solution (1 mg mL^–1, Thermo Fischer Scientific, Langenselbold, Germany). Staining was performed in the dark at room temperature for 2 min. Then, the dye was filtered off, the filter was rinsed with ultra-pure water and dried in the dark at room temperature for 5 min. Polyphosphates in the stained filaments were visualized under a fluorescence microscope (Axioskop 2 mot PLUS, Carl Zeiss, Jena, Germany) with the specific DAPI filter set (excitation: BP 390/22, beam Splitter: FT 420, emission: 460/50). Polyphosphate chains longer than 15 P-subunits formed a complex with the dye that increased its fluorescence and shifted its absorption spectrum from 456 nm – when only DNA is visualized via blue fluorescence emission – to 526 nm via bright yellow fluorescence of the accumulated polyphosphate granules (Tijssen et al., 1985 (link); Ohtomo et al., 2008 (link); Diaz and Ingall, 2010 (link)).