Tetrazolium mtt

Cells were plated at 1.4x10⁵ cells/well in a 12-well plate and transduced the next day with FcγRIa or Fluc-encoding lentiviruses as described above. Two days post-transduction, cells were infected with wild type Lm for 1 hr (MOI = 0.015, 0.05, 0.1), washed with medium, supplemented with 50 μg/ml gentamicin (Quality Biological), and then gently overlaid with 1.5ml/well of DMEM, containing with 10% FBS, 0.4% agarose, and 20 μg/ml gentamicin (Quality Biological). The overlay was allowed to stabilize for 15 min at room temperature, when plates were moved back to an incubator at 37°C. Foci of Lm infection were visualized 30 h after initial infection by adding 200μl of 5mg/ml (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (tetrazolium MTT) (Sigma) solution to each well and incubating at 37°C for 3 h. Plates were scanned and foci of infection quantified using ImageJ software.

The method is based on the reduction of the yellow tetrazole 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the mitochondrial dehydrogenases to purple formazan dye. Briefly, 5,000 cells per well were plated in 200 µl of medium on a 96-well plate. The cells were allowed to attach overnight and treated with COX-2 or NF-κB inhibitor for 72 h. For each inhibitor concentration, there were six replicate wells. A total of 20 µl of the 5 mg/ml tetrazolium MTT (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and left for 3 h in an incubator at 37°C, 5% CO₂ allowing the cells to metabolize the dye. The medium was removed and the formed crystals of purple formazan were dissolved with 150 µl isopropanol (Sigma-Aldrich, St. Louis, MO, USA). The plates were wrapped in aluminum foil and agitated on a shaker for 20 min. Absorption of the purple MTT solution was measured at 570 nm with a BioTrak II plate reader (Amersham Biosciences, supplied from Vector Scientific, UK). Cell viability was calculated after subtracting absorption of isopropanol, which was used as a blank by normalizing to the untreated control.