Fig. 2

Analyses of the glycocalyx configuration by AFM-based techniques and 3D-SIM imaging before and after treatment with a glycocalyx-targeting enzymatic cocktail. (A, B) Examples of recorded Force Curves (FCs) for the measurement of the glycocalyx thickness (following the Sokolov et al. protocol [34,35,36,37], further details can be found in Additional file 1: SI1) on control PC12 cells with their native glycocalyx, and after enzymatic treatment for the digestion of the glycocalyx (hyaluronidase II, chondroitinase ABC, heparinase III, and neuraminidase). The force is plotted as a function of the tip/membrane distance (in semi-log scale); dots represent the experimental data and solid lines the fit using the brush model, where a linear regime highlights the  entropic resistance of the glycocalyx (GC) to compression. C, D Distribution of the glycocalyx thickness obtained from the experiments (one count corresponds to one cell). E Representative close-up 3D-SIM image of the periphery of a PC12 cell (on PLL), indicating the glycocalyx brush marked with wheat germ agglutinin (WGA, pixel intensities were inverted for clarity), from a plane just above the substrate which were used for the quantification of the thickness of the glycocalyx layer. Typical whole cell images can be found in Additional file 1: SI – Fig. S3. F The boxplot below shows the mean and 95% confidence intervals of the glycocalyx thickness analysis from 43 such close-up zones taken from 15 different images. G Examples of two FCs with highlighted selected regions for linear fit, the inset shows the results of the effective glycocalyx stiffness. H, I On the left, AFM image of native PC12 cells covered with the glycocalyx (scan of the cell membrane obtained by peak force tapping technique on fixed samples) is shown (upper image), compared to the situation after enzymatic glycocalyx digestion (bottom image). On the right, the according cartoons summarise and integrate the data obtained with the AFM- and 3D-SIM-based glycocalyx analyses and visualise schematically the glycocalyx configuration in its native state and after the enzymatic treatment. These schemes were created with BioRender.com. HS = Heparan-Sulphate, KS = Keratan-Sulphate, CS = Chondroitin-Sulphate, HA = Hyaluronic acid, GC = Glycocalyx