Red blood cell ghosts

To obtain images of the cytoskeletal network and its individual constituents, RBC ghosts were formed. Components of the solution in which the RBC were stored were removed. In order to do this, we took 50 μL of PRBC mixed with 500 μL of phosphate-buffered saline (PBS) at pH 7.4 (MP Biomedicals LLC, Illkirch-Graffenstaden, France). This suspension was stirred on a mini-rotator Bio RS-24 (Biosan, Riga, Latvia) with a rotation frequency of 6–8 rpm for 5 min. This step was important for a thorough, uniform, slow RBC washout from the storage solution. The suspension was then centrifuged at 1,500 rpm for 5 min in a centrifuge Universal 320 (Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany). After removal of the supernatant fluid, 75 μL of sediment cells remained. Next, 500 μL of PBS were added. The washing process was repeated five times. The next stage was haemolysis of the RBC, which was achieved by adding 500 μL of hypotonic solution (0.09% NaCl in distilled water) to the cells. The suspension obtained was centrifuged at 3,000 rpm for 5 min. The supernatant was removed, and 300 μL of distilled water were added. Finally, the derived sample was placed in a refrigerator at 4°C for 30 min, after which the mixture was kept for 10 min at 20°C. The RBC ghosts formed in the resulting solution. The suspension obtained was centrifuged at 3,000 rpm for 5 min. The supernatant was removed and the 10 μL of sediment were used to prepare a ghost monolayer smear on a glass slide, using a V-Sampler^® device (Vision, Vienna, Austria). The ghost monolayer was scanned with an atomic force microscope (AFM). No membrane fixatives, including glutaraldehyde, were used at any stage of the preparation of the cytoskeletal mesh. The procedure used to obtain the RBC ghosts has been described in detail previously¹⁷.