Cells were seeded on 18mm coverslips coated with collagen and allowed to adhere for a minimum of 4 hours. AFM measurements were performed with an Asylum MFP3DAFM (Asylum Research, CA) coupled to a Nikon TE2000E2 epifluorescence microscope. Individual cells were indented using a pyramid-tipped probe (Olympus) with a nominal spring constant of 10 pN/nm. The probe was first calibrated in air using thermal vibration method to determine the exact spring constant. The first 1μm of force-indentation curves for individual cells were fitted with the Hertzian model for a pyramidal tip to obtain estimates of cortical stiffness. At least 100 cells across three experiments were analyzed per condition.
Pyramid tipped probe
Manufactured by Olympus
The Pyramid-tipped probe is a laboratory equipment designed for precise sample manipulation. It features a pyramid-shaped tip that allows for delicate and controlled handling of small samples or components. The probe's core function is to facilitate accurate positioning and movement of materials during various laboratory procedures.
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3 protocols using pyramid tipped probe
1
Atomic Force Microscopy of Cell Cortical Stiffness
2
Measuring Cortical Stiffness in GFP-tagged Cells
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MCF-7 cells were transfected with plasmid DNA encoding GFP-tagged NMHC-IIA, -IIB, or -IIC1. Culture dishes were mounted onto the stage of an Asylum MFP3D atomic force microscope (Asylum Research) coupled to a Zeiss epifluorescence microscope and indented using a pyramid-tipped probe (Olympus) with nominal spring constant of 20 pN/nm. GFP-positive cells were selected for the measurements, which were done in a stretch of the membrane where there was no protrusion. Actual spring constants were estimated using a thermal calibration method, and force curves were recorded for >20 cells for each sample. Force–indentation profiles were fit with a modified Hertzian model of a cone indenting a semi-infinite elastic material to extract the magnitude of cortical stiffness (MacKay and Kumar, 2013 (link)).
3
Measuring Cellular Cortical Stiffness
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CHO cells were cultured on tissue culture plates for 24 h before initiating the experiments. Culture plates were mounted onto the stage of an Asylum MFP3D AFM (Asylum Research, CA) coupled to a Zeiss epifluorescence microscope and indented using a pyramid-tipped probe (Olympus) with nominal spring constant of 20 pN/nm. Actual spring constants were determined using thermal calibration method. Force curves were obtained for 30-40 cells for each condition and each time point. Force-indentation profiles were fit with a modified Hertzian model of a cone indenting a semi-infinite elastic material to extract the magnitude of cortical stiffness46 (link).
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