Genjet in vitro dna transfection reagent for u2os cells

HeLa, HEK293T, MCF7, and U2OS cells, purchased from ATCC, were grown at 37°C and 5% CO₂ in DMEM high glucose medium (GIBCO, #11965092) supplemented with 10% FBS (Corning Cellgro, 35–010-CV), 1 mM sodium pyruvate (GIBCO, #11360070), and 1X nonessential amino acids (GIBCO, #11140076). Transient transfections were performed using GenJet In Vitro DNA Transfection Reagent ver. II (SignaGen, #SL100499), GenJet In Vitro DNA Transfection Reagent for MCF7 cells (SignaGen, #SL100489-MCF7), or GenJet In Vitro DNA Transfection Reagent for U2OS cells (SignaGen, #SL100489-OS) following the manufacturer’s instructions. For biosensor barcoding, 2×10⁵ cells were seeded in 12-well plates and allowed to attach overnight. Cells in each well were transfected with a pair of barcoding proteins and one biosensor, using a total of 0.75 μg plasmid per well. The next day, cells were harvested from each well with Accutase (Corning, 25–058-CI) and mixed together. The resulting cell mixture was then seeded into 35 mm glass-bottom dishes (Mattek, P35GC-0–14-C) at 7×10⁵ cells per dish and incubated at 37°C and 5% CO₂ overnight. The cells were starved in serum-free, phenol red-free DMEM (GIBCO, #21063029) for 1 h before imaging experiments.