Neubauer improved chamber

Spores were purified as described by [57 (link)] with some modifications. An overnight culture of B. thuringiensis in 4 ml of TSB was grown at 37°C, 180 r.p.m. The overnight culture was diluted 1:100 in 50 ml fresh TSB media containing 0.1 mmol MnSO₄ (PanReac, AppliChem) and grown at 37°C, 180 r.p.m. for 10 days. Cultures were transferred to a 50 ml falcon tube and stored on ice for 20 min. The tubes were spun down at 4°C, 10 000 r.p.m. for 10 min. The pellet was suspended in 20 ml of 50 mM Tris–HCL (pH 7.2; PanReac, AppliChem) with an addition of 50 µg ml⁻¹ lysozyme (from hen egg whites, Fluka). Samples were incubated at 37°C, 180 r.p.m. for 1 h. The sample was spun down at 4°C, 10 000 r.p.m. for 10 min, and the pellet was suspended in cold sterile water. This washing step was repeated twice. The pellet was then suspended in 5 ml 0.05% SDS solution (Sigma, D6750-10G) by vortexing and incubated for 5 min at room temperature. The sample was then spun down at 4°C, 10 000 r.p.m. for 10 min, and the pellet was suspended in cold sterile water. This washing step was repeated five times. After the final washing step, the pellet was suspended in 5 ml of cold, sterile water and stored at 4°C for later use. Spores were counted using a Neubauer-improved chamber (Paul Marienfeld GmbH, Lauda-Konigshofen, Germany) with a chamber depth of 0.1 mm.

The invasion assay was performed as previously described [69 (link)]. Essentially, 1 × 10⁵ cells were seeded into the upper well of the boyden chamber in 1000 µL medium. KAN0438757 was added in different concentrations into both sides of the chamber. After 96h incubation at 37 °C, 5% CO₂ the medium from the upper chamber was removed and the content of the lower part, containing the invaded cells (floating as well as adherent cells), was collected for each condition. Next, cells were centrifuged at 500 g, for 5 min, stained with 0.4% Trypan Blue (Carl Roth, Karlsruhe, Germany) and manually counted under the microscope using a Neubauer-improved Chamber (Paul Marienfeld GmbH, Lauda-Königshofen, Germany).

Proliferation rates of cultured on the collagen matrices hPDL and hOF cells were determined by trypan blue (Sigma-Aldrich Chemie GmbH) dye exclusion cell counting performed in a Neubauer-improved chamber (Paul Marienfeld, Lauda-Königshofen, Germany). After 24 h of starvation, 5 × 10³ cells/well were plated in 3% FCS/DMEM either on collagen matrices (test groups) or on tissue culture plastic (control group) and allowed to proliferate for 11 days. The culture media was replaced every 3 days. Viable cells of each group were counted independently and blindly by two of the co-authors on a Leica DM IL LED microscope on day 1, 3, 5, 7, 9, and 11. Data represent means ± SD from three independent experiments performed with three different cell donors for each cell type.