Metavue software

MV3 cells were seeded onto glass coverslips coated with collagen I
(0.4 mg/ml collagen diluted in PBS, from bovine calf skin; Biochrom).
Cells were fixed with 3.5% PFA in PBS for 30 min at RT. Afterwards,
cells were washed 3x in PBS, permeabilised with 0.1% Triton X-100 in PBS and
again washed 3x in PBS. Blocking occurred in 3% BSA diluted in PBS for
3 h at RT. The monoclonal mouse anti-CD166/ALCAM antibody (L50; dilution
1:100 in 3% BSA-PBS; ThermoFisher Scientific) served as primary antibody
incubated at 4 °C overnight. After the next washing procedure,
the secondary antibody (goat anti-mouse Alexa 488, dilution 1:600 in 3% BSA-PBS;
Molecular Probes, Eugene, USA) was incubated for 45 min at RT and probes
were washed again. Fluorescence mounting medium (Dako, Glostrup, Denmark)
supplemented with DAPI (Invitrogen) was used to mount the samples.
Immunofluorescence images were acquired under wide-field fluorescence conditions
with an inverted microscope (Axiovert 200, Zeiss) equipped with a
100 × 1.45 objective lens. The following filter sets
were used: excitation 570/40 nm, beam splitter 510 nm, emission
540/50 nm (for Alexa 488-fluorescence) and excitation 365/12 nm,
beam splitter 395 nm, emission 397 nm (for DAPI fluorescence).
Image acquisition was performed with a digital camera (model 9.0, RT-SE-Spot;
Visitron Systems, Puchheim, Germany) and the MetaVue software (Visitron
Systems).

Cells were seeded onto collagen type I-coated (Collagen G, Biochrom AG; final concentration 0.4 mg ml⁻¹) coverslips and cultured for 2 h. The cells were then fixed with 3.5% paraformaldehyde (w/v) in PBS (Dulbecco, Biochrom AG) for 30 min and permeabilized for 25 min in 0.1% (v/v) Triton X-100/TBS in order to ensure that the intracellular F-actin epitopes were accessible to the antibody. After washing with PBS (2×), nonspecific binding sites were blocked with 3% bovine serum albumin (BSA) in PBS (w/v) for 2 h at room temperature. The cells were then stained with Alexa Fluor® 488 Phalloidin (Invitrogen AG; dilution 1:100) for 45 min. Prior to Dako mounting (Dako A/S, Glostrup, Denmark), the cells were washed in PBS once again. Images were taken with a digital camera (Model 9.0, RT-SE-Spot, Visitron Systems, Puchheim, Germany) fitted to an inverted microscope (Axiovert 200, Carl Zeiss AG) and controlled by MetaVue software (Visitron Systems).

We measured the activation of caspase 3 in living cells after 24 h treatment using the CaspGLOW^TM Red Active Caspase-3 staining kit (BioVision Inc., USA). For each condition 3 × 10⁵ cells were seeded in a glass-bottom dish (pre-coated at 37°C with 0.1% poly-L-lysin for 30 min) and cultured overnight; the day after, individual dishes were subjected to different treatments. After 24 h of treatment, the cells were washed with PBS and then stained with Red-DEVD-FMK at 37 °C in the dark for 1 h and further processed according to the manufacturer’s protocol. Cells were kept in 500 μL of the washing buffer and fluorescent images were recorded (546 nm excitation, 590 nm emission). Images from ten fields of view for each dish were acquired using a ZEISS microscope Axiovert 200 with a digital camera (Model 9.0, RT-SE-Spot, Visitron Systems, Germany) controlled by MetaVue software (Visitron). Cellular mean fluorecence intensities were determined with ImageJ.