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. 2016 Jun 27;6:26935. doi: 10.1038/srep26935

Figure 4. Physical pressure on RBC membrane induces NO production.

Figure 4

(a) Schematic representation of physical perturbation of RBC. RBCs were pre-incubated with DAR in presence of NOS substrate (L-arginine) and NOS inhibitor (L-NAME) for 30 minutes at 37 °C. RBCs were placed between poly L-lysine coated cover slips, and placed under an inverted microscope. External weights (16 gm) were incrementally stacked on the coverslip at one minute intervals, and imaged after each interval. (b) Real time NO production was captured by fluorescence microscopy. When compared to 0 gram control, RBCs loaded with 65.15 g showed a significant increase in NO production (n = 3; *p = 0.000313). In presence of L-Arginine, RBCs loaded with all weights (apart from 16.38 gm), showed increased NO production when compared with 0 gram (control) (n = 3; **p = 0.00248; **p = 0.00035, **p = 3.77E-07, **p = 1.29E-06, **p = 0.000749). NO production by loaded RBCs in presence of L-Arginine was also greater than that observed in loaded RBCs without L-Arginine (n = 3; $p = 0.000185, $p = 0.0000186, $p = 0.00238, $p = 1.49E-06). Addition of L-NAME also decreased NO production by loaded RBCs. (c) Fluorescence images of RBC under mechanical perturbation were imaged by OlympusIX71 microscope using 60× magnification and oil-immersion objective lenses with a numerical aperture (NA) of 1.42.