Protocol for assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells-Animal

Cultivate bovine fibroblast cells at 5.0x10⁵ cells per culture flask with a 75 cm2 surface area. Well area coverage, between 60-80% confluence. It takes approximately 8-10 days.

Before starting dissociation, place the working aliquot of 0.05% trypsin and EDTA solution in the incubator at 37°C for at least 0h 10m 0s.

Remove the DMEM culture medium and wash with Dulbecco's Phosphate-Buffered Saline (DPBS, GIBCO). Discard the DPBS and immediately add 2mL of 0.05% trypsin and EDTA. Make sure to cover the plate area.

Place the culture flask in the incubator at 37°C for 0h 5m 0s.

Add 2mL of DMEM.

Transfer the loose cell suspension to a 1.5 or 2 mL centrifugation tube.

Spin at 1300rpm.

Aspirate and discard the supernatant.

Wash with 5mL of DPBS using a serological pipette.

Spin at 1300rpm.

Aspirate and discard the supernatant.

Resuspend the cell pellet with fresh DMEM medium using a serological pipette.

Add 1mL of cells/flask culture to new culture flasks with a 75 cm² surface area that have been previously prepared.

Incubate the cells at 37°C, 95% humidity, 5% CO₂.

After 24h, replace the old culture medium with 10mL of fresh DMEM medium.

Divide the cells every 5 days.

Prepare the cells for transfection: the cells should be at a specific stage of culture and in the appropriate medium for transfection.

Prepare the transfection solution: mix the LTX Plus with the desired plasmid following the manufacturer's instructions.

Add the transfection solution to the cells: add the transfection solution to cells grown in culture plate or to the medium for suspended cells.

Incubate the cells: incubate the cells with the transfection solution for a specific period of time, usually 4-6 hours.

Dissociating cells using 0.05% Trypsin-EDTA (1X):

Remove the old growth medium from each well and wash with 0.5 mL/well of DPBS, remove immediately.

Add 0.5mL/well of 0.05% Trypsin-EDTA.

Incubate the cells in the incubator at 37°C for 0h 5m 0s.

Add 0.5mL/well of DMEM in a microcentrifuge and 1.5 or 2 mL tube containing DMEM.

Spin at 1300rpm.

Wash the cells with DPBS.

Spin at 1300rpm.

Remove the supernatant and resuspend the cells with DPBS.

Transfer the cells to a 1.5 mL centrifugation tube for flow cytometry analysis.

Count the cells with a Neubauer chamber and cultivate bovine fibroblasts and HEK 293T cells in 96-well plates at a density of 1x105 cells/well in triplicate and grown for 24h 0m 0s at 37°C in a 5% CO₂ atmosphere.

After 48 hours of transient cotransfection, incubate the cells with 15µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Thermo Fisher Scientific) (5 mg/mL).

Incubate for 4h 0m 0s at 37°C.

Remove the MTT solution and add 150µL of DMSO to each well to dissolve formazan crystals.

Read the absorbance at 595 nm in a plate reader or spectrophotometer.