96 well cell transformation assay

Adhesion independent growth was assessed using Cell Biolabs Inc 96 Well Cell Transformation Assay (Cell Biolabs; Cat#: CBA-135). Transduced or untransduced cells were plated at a density of 3,000 cells per well in a 48 well dish and were suspended in 150ul agar/media solution. This cell quantity, well size, and volume of agar/media were constant for both minilibrary screening and targeted mutagenesis assays. Six replicated were plated following single viral transduction for targeted mutagenesis assays. Transformation was monitored for one week, and colonies were collected following the manufacturer’s instructions. In short, Cell Biolabs Inc matrix solubilization solution was added to each well of cells (Cell Biolabs; Cat#: CBA-135). Colonies were resuspended in OSE media and plated at very low density on 15cm plates. Individual, distinct colonies growing on 15cm plates were picked and cultured independently. Sterile filter paper was soaked in trypsin and was used to pick individual colonies from plates. Picked colonies were added to 24-well plates and were passaged using OSE culturing methodology. Brightfield images of culture wells were taken using a Nikon SMZ1500 microscope and Nikon Digital Sight DS-Fi1 camera system at 2x magnification. 10x and 20x images were taken using a Nikon TMS-F microscope and Moticam 2300 3.0MP Live Resolution camera system.

HaCaT cell was irradiated with 3 mJ/cm² UVB once every other day for three weeks to generate UVB-induced transformed HaCaT cell line. The success of cell transformation was determined by 96-well cell transformation assay following the manufacture protocol (Cell Biolabs, Inc.). Briefly, base agar layer was prepared using 1.2% agar solution with 2XDMEM/20%FBS and solidified at 4 °C for 30 minutes. The cell agar layer was prepared similarly, with 5000 cells/well seeded in each well of a 96-well plate. 100 μL of cell culture medium was added into each well, and the plate was kept in the incubator for 10 days at 37 °C and 5% CO₂. For cell harvest, 50 μL of agar solubilization solution was added to each well and incubate for 1 h at 37 °C, followed by 25 μL lysis buffer to each well. Cells were stained with CyQuant GR Dye and the fluorescent was read at 485/520 nm.