Atomic Force Microscopy (AFM)

An AFM (JPK NanoWizard® 4 XP, Bruker, Germany) coupled with an inverted Olympus IX73 optical microscope was used to investigate the mechanical properties of living T24 cells upon treatment with BFA (20nM), TG (1-100nM), CytD (100nM), Wortmannin (1µM) as well as co-incubation of BFA and TG with CytD. Cells were seeded in tissue culture dishes (REF: 40 93040, TPP Techno Plastic Products AG, Switzerland) for 24–48 h for T24 and 48–72 h for fibroblasts to obtain cell density of 70–80%. After this period, cells were incubated with the respective treatments for 2 to 24 h. Following incubation, cells were washed once with pre-warmed LCIS and subsequently imaged in LCIS using QI™ mode and PFQNM-LC-A-CAL AFM tips (Bruker) with calibrated spring constant k ranging from 0.125 N/m to 0.144 N/m. QI™ settings used are the following: Z-length: 1000 nm; applied force 0.2 nN; speed 100 µm/s [44]. Living cells were randomly selected using the optical microscope and imaged in a 25 µm × 25 µm square area. Cells were imaged for a maximum of 60 min to ensure stable cell viability. In total, 13 to 32 cells for each experimental condition derived from at least 3 independent cell preparations (biological replicates) were imaged and force curves were processed to obtain data of the Young´s modulus. The raw data was processed using JPK NanoWizard® Data processing software. Within the images, an area of 3.1 µm × 3.1 µm was selected in the nuclear, perinuclear or cytoplasmic region. A representative force curve of the cell was chosen to set up the processing of the Young’s modulus. First, through the calculation of the average value of a section in the baseline and subsequent subtraction of the whole curve, the baseline offset in vertical deflection was removed. Then, the vertical deflection was plotted against the vertical tip position. Afterwards, the reference force height was determined at the height value at 50% of the applied setpoint force. Finally, elasticity fit was used to calculate the Young´s modulus. Here, the Hertz/Sneddon fit [45, 46] was used for a paraboloid tip shape with a 70 nm tip radius and a Poisson´s ratio value of 0.50 [47, 48]. Force curves were batch-analyzed in the selected area of the nuclear, perinuclear, and cytoplasmic regions and median values of the Young´s modulus were calculated [49].