The reporter cell line has been cultured following the suppliers recommendation (see below):

• Thaw Medium 1 (BPS Bioscience #60187): MEM medium (Thermo Fisher, #11095098) supplemented with 10% FBS (Thermo Fisher, #26140079), 1% non-essential amino acids (Corning, #25-025-CI), 1 mM Na pyruvate (Corning, #25-000-CI), 1% Penicillin/Streptomycin (Thermo Fisher, #15140163)
• Growth Medium 1B (BPS Bioscience #79531): Thaw Medium 1 (BPS Bioscience #60187) + 400 µg/ml of Geneticin (Invitrogen #11811031).

Cell Culture Protocol Cell Thawing

1. To thaw the cells, it is recommended to quickly thaw the frozen cells from liquid nitrogen in a 37°C waterbath, transfer to a tube containing 10 ml of Thaw Medium 1 (no Geneticin), spin down cells at 1000 rpm, resuspend cells in 5 ml of pre-warmed Thaw Medium 1 (no Geneticin), transfer resuspended cells to T25 flask and culture at 37°C in a 5% CO2 incubator overnight.

2. The next day, replace the medium with fresh warm Thaw Medium 1 (no Geneticin), and continue growing culture in a CO2 incubator at 37°C until the cells are ready to be split.

3. Cells should be split before they reach complete confluence.

4. At first passage switch to Growth Medium 1B (contains Geneticin).

Cell Passage

1. To passage the cells, rinse cells with phosphate buffered saline (PBS), detach cells from culture vessel with 0.05% Trypsin/EDTA.

2. After detachment, add Growth Medium 1B (contains Geneticin) and transfer to a tube, spin down cells, resuspend cells in Growth Medium 1B (contains Geneticin) and seed appropriate aliquots of cell suspension into new culture vessels.

After passing, the cells are quite sparse and growing really slowly. I am open to any recommendations.

Many thanks in advance .

BW

Nimo