Hacat Cell Information and Protocols

HaCaT Cell Information and Protocols

· The HaCaT cell line is a well-known immortalised human keratinocyte cell line.

· Use DMEM media (see DMEM protocol) with 10% FCS and 1% P/S.

· Cell doubling time is approximately 24 hours (you can use this to calculate dilutions for splitting cells)

· Media should be renewed twice weekly and cultures split when 80% confluency is reached

Splitting HaCaT Cells

· Remove media from flask and add 10ml PBS to rinse cells then remove;

· Add 2-3ml trypsin and leave until all cells have rounded up and begun to detach (for ~10min) in 37oC/5% CO2 incubator;

· Bang flask to dislodge cells (If cells are not sliding down flask easily then place flask back in incubator for a further 1-2min and try again);

· Add 10ml DMEM/10% FBS media to flask and pipette up and down to remove clumps and resuspend cells;

· Transfer to a 15ml Falcon tube and centrifuge @ 1,500 rpm for 5min with a balance;

· Pour off supernatant and resuspend in approximately 10ml of fresh media;

· Transfer required amount (this will depend on how long you wish to grow them for) into a new flask and add fresh media so final volume in the flask is between 20 and 25 mL.

And/or

· Perform a cell count on cell suspension and adjust to required cell concentration with fresh media to perform assay or experiment.