Syto 59

For immunostaining cells were cultivated on glass bottom dishes (MatTek, El Segundo, CA, USA). Astrocytes were fixed in 4% paraformaldehyde for 15 min. Then cells were washed once in 120 mM Na2HPO4 for 10 min, once in low-salt buffer (150 mM NaCl, 10 mM Na2HPO4) for 10 min and twice with high-salt buffer (0.5 M NaCl, 20 mM Na2HPO4) for 10 min. Cells were permeabilized and blocked with FSBB buffer (0.1% Triton X-100, 5% FBS in PBS) for 20 min and incubated overnight at +4 °C with primary anti-GFAP antibody (Sigma-Aldrich, St. Louis, MO, USA) (1:100 in FSBB), to detect astrocytes. The following day, the cells were washed once with low-salt buffer for 10 min, twice with high-salt buffer for 10 min and incubated with secondary antibody (AlexaFluor, (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA) (1:500 in FSBB) for 1.5 h at room temperature. Then the cells were washed once in 120 mM Na2HPO4 for 10 min, once in low-salt buffer (150 mM NaCl, 10 mM Na2HPO4) for 10 min, twice with high-salt buffer (0.5 M NaCl, 20 mM Na2HPO4) for 10 min and incubated with the DNA-tropic dye Syto-59 (Sigma-Aldrich, St. Louis, MO, USA) (1:20,000 in 5 mM Na2HPO4) for 20 min. The cells were washed three times with 5 mM Na2HPO4 to remove the excess dye. Images were captured using a confocal laser-scanning microscope (LSM 700, Carl Zeiss, Oberkochen, Germany).

Material from the upper test tube volume and the test tube bottom of 14-day old cultures (#49134 and MH strain) was removed by gentle suction using a cut-off 1 mL pipette tips (to minimize shearing), and labeled with LIVE/DEAD BacLight (Invitrogen, Carlsbad, CA, USA) according to the manufactures instructions. In the same way, more 14-day old cultures were stained with Concanavalin A (25 µg mL⁻¹, (ConA, Vector Laboratories, Inc., Burlingame, CA, USA) and Syto59 (5 µM, Invitrogen, Carlsbad, CA, USA), or Wheat Germ Agglutinin (25 µg mL⁻¹) (Vector Laboratories, Inc.) and Syto59, or Sypro Orange and Syto59, or Nile Red (5 µg mL⁻¹, Sigma-Aldrich Corp. St. Louis, MO, USA), or Thioflavin T (20 mM, Sigma-Aldrich). Fluorescence in situ hybridization (FISH) was performed on material collected from the test tube bottom, which was hybridized with the EUB338-Cy3 probe [34] (link) or the NONEUB-Cy5 probe [35] (link) at a final concentration of 5 ng µL⁻¹ for 90 min at 46°C. These specimens were counter-stained with Syto59. All samples were examined either with a Leica DM RXE microscope with a TCS SP2 AOBS confocal system (Leica Microsystem, Exton, PA, USA) or a LSM710 (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA.) using confocal and transmitted imaging with the appropriate laser wavelengths lines and detection windows.