μclear base

SiMa cells (DSMZ) were grown in RPMI media (Life Technologies) supplemented with 10% Fetal Bovine Serum (FBS) (Life Technologies). SH-SY5Y cells (Sigma) were grown in a 1:1 mix of MEM (Life Technologies) and F12 nutrient mix (Life Technologies) supplemented with 15% FBS and 1% non-essential amino acids (NEAA) (Life Technologies). Cells were maintained at 37°C, 5% CO₂. Cell lines were frozen after receiving from supplier and were not used for more than 20 passages (10 weeks).
For differentiation, plates were pre-coated with 10 μg/ml laminin (Sigma) and left at 37°C for at least one hour before washing twice with PBS. SiMa cells were seeded at a density of 1 x10⁴ cells per well in 96-well plates or 2 x10⁴ cells per well in 48-well plates and incubated for 72 hours in differentiation medium (RPMI, 1x B27 (Life Technologies), 1 mM HEPES (Fisher) and 1% NEAA) with or without 10 μM AT-RA (Sigma). SH-SY5Y cells were seeded at 5 x10³ cells for 96-well plates and 2 x10⁴ cells for 48-well plates. They were differentiated for 144 hours, using the same differentiation media as SiMa cells, before treatment. 96-well plates with a μClear base (Greiner Bio-one) were used for microscopy.

Two hundred and fifty micro liter of a mid-exponential phase culture (10⁹ CFU/ml) was added to the wells of a polystyrene 96-well microtiter plate (Greiner Bio-one, France) with a μclear^® base (Polystyrene, thickness of 190 μm ± 10%) which allowed high resolution confocal imaging. After 1 h of adhesion at 30°C, the wells were refilled with 250 μl MRSm. This preparation was then subjected to Confocal Laser Scanning Microscopy.

Cell lines were maintained in a 37 °C incubator at 5% CO₂. The N2a (ATCC), J774.2 (ECACC) and A549 (ECACC) cell lines were cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). SH-SY5Y cells (Sigma-Aldrich) were cultured in a 1:1 ratio of Eagle’s Minimum Essential Medium (Gibco) and F12 Medium (Gibco) supplemented with 1% non-essential amino acids (Gibco) and 15% FBS. HeLa cells (ECACC) were cultured in EMEM with 10% FBS, U937 cells (gift from Dr Jim Gallagher) were cultured in RPMI (Gibco) with 25 mM HEPES (Gibco) and 10% FBS, and RBL-2H3 cells (gift from Dr Birgit Helm) were cultured in DMEM (Gibco) with 1% non-essential amino acids and 10% FBS. Cells were plated at a density of 3 × 10⁴ cells per well in uncoated 48-well plates (Corning) or at 5 × 10³ cells per well in uncoated 96-well plates (Corning). For microscopy, 96-well plates with a μClear base were used (Greiner Bio-one) for better resolution.