Ti eclipse inverted widefield fluorescence microscope

Immunocytochemistry was used for visualization of actin stress fibers and vinculin focal adhesions. Cells were exposed to the different stress medium and gravity conditions as described in “Experimental procedures”. Three out of six replicates were processed for immunocytochemistry analyses of the cytoskeleton. Fixed cells were incubated in PBS containing 0.1% Triton-X 100 and 3% BSA for 5 min at room temperature and washed with Tris buffered saline containing 0.1% Tween (TBS-T). Blocking was performed with 5% goat serum in Tris-NaCl-blocking buffer (TNB) for one hour at room temperature. Primary antibody to stain vinculin focal adhesions (mouse monoclonal anti-vinculin (7f9), Santa Cruz, sc-73614) at a dilution of 1/500 in TNB was added and samples were incubated overnight at 4 °C. Afterwards, samples were washed in TBS followed by incubation with the secondary antibody (Alexa Fluor goat-anti-mouse 488, Invitrogen A11001, 1/500) and Phalloidin 594 (actin stress fibers) TRITC (Invitrogen A12381) in TNB for two hours at room temperature in the dark. Counterstain was carried out using DAPI (1 µg/mL). Immunofluorescence-stained cells were visualized using a Nikon Ti Eclipse inverted widefield fluorescence microscope (Nikon Instruments) with ×60 objective and immersion oil. z-stacks of 11 images were taken 0.5 µm apart.

For each condition and at each time point (30 min, 1 h, 4 h, and 48 h), four wells were used as technical replicates for immunocytochemical visualization of DNA DSBs. Fixed cells were incubated in PBS containing 0.25% Triton X-100 for three minutes at room temperature. After rinsing with PBS, samples were blocked with Tris-NaCl-blocking buffer (TNB) containing 5% goat serum for one hour at room temperature. Primary antibodies (mouse monoclonal to γ-H2AX, Millipore 05–636, at 1/300 and rabbit polyclonal to 53BP1, Novus Biological NB 100–304, at 1/1000) were diluted in TNB and added to the samples for one hour at 37 °C. Samples were washed three times with PBS and incubated with TNB containing secondary antibodies (Alexa Fluor 488 goat-anti-mouse, 1/300 and Alexa Fluor 568 goat-anti-rabbit, 1/1000) for one hour at 37 °C. Cells were rinsed and mounted with IBIDI mounting medium containing DAPI (IBIDI, 50,011). A Nikon Ti Eclipse inverted wide field fluorescence microscope (Nikon Instruments) with a 20 × objective connected to a Prime BSI sCMOS camera was used to visualize cell nuclei, γ-H2AX and 53BP1 foci. Per well, images at four locations were obtained with z-stacks of 11 images that were taken 0.9 µm apart.

For each condition, three out of six fixed scratched samples were used for immunocytochemical visualization of actin stress fibers and vinculin focal adhesion complexes. Fixed cells were incubated in PBS containing 0.1% Triton-X 100 and 3% bovine serum albumin (BSA, A2153n Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 5 min at room temperature, after which they were washed with Tris-buffered saline containing 0.1% Tween (TBS-T). For blocking, a solution of 5% goat serum in Tris-NaCl-blocking buffer (TNB) was used for one hour at room temperature. Primary antibody (mouse monoclonal anti-vinculin (7f9), Santa Cruz, sc-73614) at dilution of 1/500 in TNB was incubated overnight at 4 °C. Samples were washed with TBS and incubated with TNB containing secondary antibody (Alexa Fluor goat-anti-mouse 488, Invitrogen A11001, 1/500) and Phalloidin 594 TRITC (Invitrogen A12381) for actin stress fibers. Slides were mounted with mounting medium containing DAPI (Molecular Probes Prolong Diamond Antifade Mountant, p36962). A Nikon Ti Eclipse inverted widefield fluorescence microscope (Nikon Instruments) with a 60× objective and immersion oil and connected to a Prime BSI sCMOS camera was used to visualize the cellular components of isolated cells that had migrated into the open wound area. Z-stacks of 11 images were taken at 0.5 µm apart.