Varioscan multimode microplate reader

The quantity of VEGF A, present in the supernatant of MCF7 cells with depleted FRG1 and MDA‐MB‐231 cells with ectopic FRG1 expression, was measured using the Human VEGF Quantikine ELISA Kit (R&D Systems, MN, USA). Briefly, 1 × 10⁶ cells were plated into a 100 mm dish in complete cell culture media. On the next day, the entire media was replaced by the serum‐free media and incubated for the next 24 h. Subsequently, the supernatant was collected and centrifuged at 1792 g for 10 min at 4 °C to eliminate the debris. This supernatant was used to carry out the ELISA as per the manufacture's (R&D Systems, Minneapolis, MN, USA) instruction. OD value was taken at 450 nm in the Varioscan multimode microplate reader (Thermo, Waltham, MA, USA).

Transfected RAW macrophage cells with TNT-pEGFPC1 and IFT-pCMV4_nn constructs were analysed after 48 h of transfections to determine TNT-induced macrophage cell death. To assess effect of small molecules on cell viability, RAW macrophage cells were treated for 48 h with different concentrations of small molecules 8, 9 and 10 (1.5 µM, 3 µM, 6 µM, 12 μM, 25 µM). MTT assay was conducted to determine cell viability using MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich]. Viable cells reduce MTT into dark blue farmazon crystals. Cells were incubated with MTT solution (0.5 mg ml^-1 in PBS) for 3 h at 37 °C. Media was removed after incubation and farmazon crystals were dissolved by addition of 100 µl of DMSO to each well. Absorbance reading at 570 nm was taken using Varioscan multimode microplate reader (Thermo scientific). Percent cell viability was calculated by relative reduction in absorbance values of treated samples compared to the control.

GM-CSF promoter (accession no. P04141) was cloned into the pGL4.23 [luc2/minP] (Promega, WI, USA) vector. Appropriate cells were plated and transfected with 990 ng of pGL4.23_GM-CSF promoter construct and 10 ng internal control pGL4.74 [hRluc/TK] (Promega, WI, USA) using Lipofectamine 3000 (Invitrogen). After 48 h, cells were harvested using the lysis buffer provided with the Dual-Glo Luciferase assay kit (Promega, WI, USA). Firefly and renilla luminescence signal was quantified using Varioscan multimode microplate reader (Thermo Scientific). For each sample, firefly luciferase activity was normalized to renilla luciferase activity.