For determination of the eGC thickness, the AFM nanoindentation technique was used as described previously [11 (link),25 (link),38 (link)]. Briefly, cells were analyzed in HEPES-buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 10 mM HEPES) supplemented with or without 1% FCS, respectively, or albumin 45 µg/mL at 37 °C in a fluid chamber with a Nanoscope V Multimode AFM (Veeco, Mannheim, Germany). The addition of slight amounts of plasma protein to the buffer is necessary for eGC preservation in vitro, as the work of our group [16 (link)] and others [27 (link)] has repeatedly demonstrated. Based on a significant amount of preliminary laboratory work with FCS and albumin, we could conclude that those are comparable in this respect. Incubation time of all AFM experiments was 60 min or as otherwise stated in the figure legend. A triangular cantilever (Novascan Technologies, Boone, NC, USA) with a mounted spherical tip (diameter 10 µm) and a spring constant of 10 pN/nm was used to periodically indent the cells. A laser beam was used to quantify the cantilever deflection. Knowing the force acting on the cantilever, the piezo displacement, and the deflection sensitivity, the thickness of the eGC could be calculated.
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