96 well plate

Cellulose production in colony biofilms was quantified by measuring the cellulose bound to cells [27 (link)]. STm WT, ∆csgD, ∆csgA, ∆bcsA, ∆csgA: ∆bcsA were spotted on LB-NaCl Agar supplemented with 2% Calcofluor white stain (Sigma Aldrich), incubated for 3 days at 25 °C. The colony biofilms formed were retrieved, mixed with 1 mL PBS, and centrifuged at 5000 rpm for 10 min to eliminate the unbound cells and Calcofluor. Cells were resuspended in water and transferred to a 96 well plate (Tarsons). Fluorescent measurements corresponding to biofilm bound cellulose (excitation 360 nm, emission 460 nm) were made in Microplate reader (Biotek). The results were analysed using GraphPad Prism5 for Windows.

Cytotoxicity was assessed by the MTT assay based on the reduction of MTT by mitochondrial dehydrogenases of viable cells to a purple formazon product^{65 (link)}. Cells were seeded in a 96-well plate (Tarsons, India) at a density of 1 × 10⁵ cells per well. After overnight growth, cells were treated with various concentrations of nimbolide (0–10 μM) and incubated for 24 h. In addition, SCC131 and SCC4 cells individually pre-treated with 3-MA (10 mM) and CQ (25 µM) followed by treatment with or without nimbolide for 24 h. 10 μL of MTT was added to each well, and the plates were incubated for 3 h at 37 °C. Then 100 μl of acidified isopropanol was added to dissolve MTT-formazon product and the absorbance was measured at 595 nm in a microtiter plate reader.