Human foreskin fibroblast
(HFF) cells
CCD-1070Sk (ATCC, CRL-2091) were cultured in DMEM/F-12 medium (Gibco,
cat no. 21041-025) in the absence of phenol red and supplemented with
10% fetal bovine serum (FBS, Gibco). The cells were grown at 37 °C
in 5% CO2. The transient transfection of Paxillin-pEGFP
(Addgene, cat. no. 15233)36 (link) was performed
using a Lipofectamine 3000 Transfection kit (Invitrogen) according
to the manufacturer’s specifications.
Cultured cells
were washed with phosphate-buffered saline (PBS) (D-PBS(−),
Nacalai tesque), and all adherent cells were detached from the culture
dish with 0.25% Trypsin-EDTA (Nacalai tesque). The cell solution was
centrifuged at 300×g for 3 min and resuspended in DMEM/F-12 medium
at 106 mL–1. HFFs were plated and incubated
at a density of 1 × 106 mL–1 on
a functionalized glass bottom dish at 37 °C and 5% CO2 for 1.5 h. An oxygen scavenger system (1 mM Trolox, 2.5 mM protocatechuic
acid (PCA), and 30 nM protocatechuate-3,4-dioxygenase (PCD)) was added
to the medium, and the adherent cells were imaged. All measurements
were made within 2 h of plating the cells.
(HFF) cells
CCD-1070Sk (ATCC, CRL-2091) were cultured in DMEM/F-12 medium (Gibco,
cat no. 21041-025) in the absence of phenol red and supplemented with
10% fetal bovine serum (FBS, Gibco). The cells were grown at 37 °C
in 5% CO2. The transient transfection of Paxillin-pEGFP
(Addgene, cat. no. 15233)36 (link) was performed
using a Lipofectamine 3000 Transfection kit (Invitrogen) according
to the manufacturer’s specifications.
Cultured cells
were washed with phosphate-buffered saline (PBS) (D-PBS(−),
Nacalai tesque), and all adherent cells were detached from the culture
dish with 0.25% Trypsin-EDTA (Nacalai tesque). The cell solution was
centrifuged at 300×g for 3 min and resuspended in DMEM/F-12 medium
at 106 mL–1. HFFs were plated and incubated
at a density of 1 × 106 mL–1 on
a functionalized glass bottom dish at 37 °C and 5% CO2 for 1.5 h. An oxygen scavenger system (1 mM Trolox, 2.5 mM protocatechuic
acid (PCA), and 30 nM protocatechuate-3,4-dioxygenase (PCD)) was added
to the medium, and the adherent cells were imaged. All measurements
were made within 2 h of plating the cells.