Figure 1.
Representative force-indentation curves from AFM and sketches of the tip geometry.
(A) The dotted and solid lines represent distinct traces of the approach of the AFM tip, as measured from samples with different physical properties. Initially, the AFM tip was located at the designed position over the sample. As the AFM tip start to approach the sample, there was no interaction force (Part I). After the AFM tip contacted with the sample at the contact point (shown by black arrow), further indentation generates the indentation depth. Constant force generates a greater indentation depth on the softer cell (Part III) than on the stiffer cell (Part II). (B) Tips with three different geometries were used in this study.
Figure 2.
The effect of AFM tip shape, indenting force at the same loading rate, and operating temperature on the effective Young’s modulus of cells.
To determine the effect of the AFM tips, (A) NIH3T3 cells and 7-4 cells were plated on type I collagen (COL I)-coated glass slides overnight. The effective Young's moduli (Eeff) of cells were measured by Bio-AFM with a pyramidal tip (labeled as P in (A)), flat tip (labeled as F in (A)), and 5 µm-bead-modified tip (labeled as B in (A)) with a 1 nN indenting force at 1 µm/sec approach velocity. To determine the effect of the indenting force at the same loading rate, cells were plated on COL I-coated glass slides overnight. The Eeff of cells were measured by Bio-AFM with a (P) pyramidal tip, (F) flat tip, and (B) 5 µm-bead-modified tip with different indenting force (0.2, 0.5 or 1 nN). To evaluate the effect of operating temperatures, (E) NIH3T3 cells were plated on COL I-coated glass slides and cultured in DMEM at 31°C, 37°C, and 43°C and in CO2-independent medium (CO2-IDM) at 31°C and 37°C. The results were expressed as the mean ± SEM by scatter dot plot. Gray symbols represent the detailed experimental data. ***p<0.001; **p<0.01; *p<0.05; N.S, no significance.
Figure 3.
The effect of culture conditions on the effective Young’s moduli of cells.
NIH3T3 cells were plated onto COL I-coated glass slides and culured in DMEM (or CO2-IDM) supplemented with or without FBS overnight. The incubating temperatures were set at room temperature (31°C), 37°C, and 43°C. (A) The cells were cultured in DMEM or CO2-IDM supplemented with or without 10% FBS at 37°C, respectively. The effective Young’s moduli (Eeff) of cells were assessed by the Bio-AFM. The results are showed in scatter dot plot by mean with standard error (SE). **p<0.01; *p<0.05; N.S., no significance. (B) The representative Max XY projection images of cells cultured in various conditions. Actin cap fibers in the apical region of the cell were re-colored green, the stress fibers in the middle region of cell were colored red, and the stress fibers in the the basal region of cell were re-colored blue. (Scale bar = 10 µm).
Figure 4.
The effect of various substrates on the effective Young’s moduli of cells.
(A) NIH3T3 cells were plated onto glass slides coated with various substrates (PLL, poly-L-Lysine; FN, fibronectin; COL I, type I collagen; COL IV, type IV collagen; GEL; gelatin) overnight. (B) To evaluate the effect of substrate concentration on the effective Young’s moduli (Eeff) of cells, cells were plated onto glass slides coated with COL I of various concentrations (50, 100, or 1000 µg/ml). (C) To evaluate the effect of substrate compliance, cells were plated onto culture dish (C), collagen gel-coated dish (Co), and collagen gel (G). The Eeff of cells were assessed by Bio-AFM. The results were expressed as the mean ± SEM by scatter dot plot. Gray symbols represent the detailed experimental data. ***p<0.001; **p<0.01; N.S, no significance. (D) Organization of actin filament in the apical actin and basal actin in NIH3T3 cells plated on different substrate (glass, PLL, FN, COL I, COL IV, GEL, Co, G). (Scale bar = 10 µm).
Table 1.
The effective Young’s moduli of NIH3T3 cells measured at different indenting force at the same loading rate.
Table 2.
The effective Young’s moduli of 7-4 cells measured at different indenting force at the same loading rate.
Figure 5.
The effect of culture passage number on the effective Young’s moduli of MKPC.
(A) MKPC at the 20th, 30th, 50th, and 90th passages were plated onto type I collagen- or fibronectin-coated glass slides overnight. The results were expressed as the mean ± SEM by scatter dot plot. Gray symbols represent the detailed experimental data. (***p<0.001; *p<0.05) (B) MKPC at the 20th, 30th, 50th, and 90th passages were stained and represented as the maximal section of confocal immunofluorescence images of β-actin (red) and α-tubulin (green). (Scale bar = 10 µm).
Figure 6.
The effect of plating density on the effective Young’s moduli of cells.
(A) NIH3T3 cells and (B) MDCK cells were plated onto COL I-coated glass slides at densities of 5, 50, or 500 cells/mm2 overnight. The effective Young’s moduli (Eeff) of cells were assessed by Bio-AFM. The results were expressed as the mean ± SEM by scatter dot plot. Gray symbols represent the detailed experimental data. (***p<0.001; N.S, no significance) (C) NIH3T3 cells were plated onto COL I-coated glass slides at densities of 5, 50, or 500 cells/mm2. The immunofluorescence results are represented as F-actin (red) and nuclei (blue). (D) AFM surface topological images in living MDCK cells and confocal immunofluorescence images of F-actin (red), α-tubulin (green), and the nucleus (blue) in stained MDCK cells that were cultured at densities of 5, 50, or 500 cells/mm2, respectively. (Scale bar = 10 µm).