The HaCaT cell lines
were maintained in 75 cm2 culture flasks containing complete
medium (DMEM with 10% heat-inactivated FBS and 1% antibiotics [100
U/mL penicillin, 100 μg/mL streptomycin, and 250 μg/mL
fungizone]) at 37 °C in a humidified incubator set at 5% CO2. Cells were subcultured once a confluence of 70–80%
was reached. Cultures were rinsed with PBS and chemically detached
using trypsin–EDTA (0.25% trypsin containing 0.01% EDTA) for
10 min. Cells were harvested and trypsin-deactivated using a complete
medium. Cells were centrifuged at 200 RCF for 5 min using a Sigma
3K15 centrifuge (Sigma, Germany). The supernatant was discarded, and
the pellet of cells is resuspended in 1 mL of 10% FBS-supplemented
medium. A 1:9 dilution of cells was made with 0.1% trypan blue, and
the cells were counted using a hemocytometer. The cell concentration
was adjusted to 100 000 cells/mL.
were maintained in 75 cm2 culture flasks containing complete
medium (DMEM with 10% heat-inactivated FBS and 1% antibiotics [100
U/mL penicillin, 100 μg/mL streptomycin, and 250 μg/mL
fungizone]) at 37 °C in a humidified incubator set at 5% CO2. Cells were subcultured once a confluence of 70–80%
was reached. Cultures were rinsed with PBS and chemically detached
using trypsin–EDTA (0.25% trypsin containing 0.01% EDTA) for
10 min. Cells were harvested and trypsin-deactivated using a complete
medium. Cells were centrifuged at 200 RCF for 5 min using a Sigma
3K15 centrifuge (Sigma, Germany). The supernatant was discarded, and
the pellet of cells is resuspended in 1 mL of 10% FBS-supplemented
medium. A 1:9 dilution of cells was made with 0.1% trypan blue, and
the cells were counted using a hemocytometer. The cell concentration
was adjusted to 100 000 cells/mL.