Nucblue readyprobe

We used standard immunofluorosence staining to observe the actin filaments and nuclei of the cells seeded on the PDMS membrane before and after stretching. To this end, the cells were first fixed with 4% paraformaldehyde (PFA) for 30 min, followed by washing with HBSS (3×). Next, the cells were stained with ActinGreen^TM 488 (Thermo Fisher Scientific) and NucBlue^TM ReadyProbe^TM reagents (Thermo Fisher Scientific) and kept for incubation at room temperature for 30 min (based on the manufacturer’s recommendations). Finally, the stained cells were washed with HBSS (3×).

The cells were fixed by treating them with 4% paraformaldehyde (PFA) for 15 min on the membrane. Subsequently, the cells were washed three times with PBS. Standard immunofluorescence staining was employed to examine the actin filaments and nuclei of the cells on the PDMS membrane before and after stretching. The fixed cells were stained with ActinGreen^TM 488 (Thermo Fisher Scientific) and NucBlue^TM ReadyProbe^TM reagents (Thermo Fisher Scientific) and incubated at room temperature for 30 min. Subsequently, the cells were washed three times with PBS. The membrane was then stored at 4 °C for subsequent imaging and analysis. The actin fibers and cell nuclei were observed using an inverted microscope (Nikon Eclipse Ti2; Nikon, Tokyo, Japan), as described in the following section.