Red 233 microscope

The
incubation was carried out as follows: RBC was fixed in the mixture
of 5% paraformaldehyde (PFA) and 0.01% glutaraldehyde (GA) for 60
min at room temperature. After fixation, cells were washed by exchanging
the supernatant with PBS buffer, settled on poly-l-lysine-treated
(0.1 mg/mL, 10 min, room temperature) cover glasses (15 min, room
temperature), and mounted 80% glycerol. The coverslips were sealed
with nail polish. Many cells in several separate experimental samples
were studied using a RED-233 MOTIC microscope (63× objective,
10× ocular). Images were acquired using a Motica 3.0 MP microscopic
camera and the program Motic Images Plus 3.0. The shapes of RBC in
every sample were estimated according to the Bessis classification.⁴¹

The absorption spectra of hemoglobin were scanned between the visible range from 450 to 700 nm in BioMate™ 160 UV–Vis spectrophotometer, using supernatants obtained from selected samples obtained in the section “Inhibition of oxidative stress-induced hemolysis”. RBC obtained from selected samples in the section “Inhibition of oxidative stress-induced hemolysis” were used for intracellular visualization of Heinz bodies. Cells were stained with methyl violet (0.5% in 0.9% NaCl) for 45 min at RT. Following incubation, RBC were washed and fixed in 5% PFA plus 0.01% GA for one hour at RT. Fixed RBC were washed by exchanging supernatant with PBS. After washing, RBC were settled on poly-l-lysine-treated (0.1 mg/mL, 10 min, RT) cover glasses and mounted on 80% glycerol. The cover slips were sealed with nail polish. A large number of cells in several separate experimental samples were studied using a RED-233 MOTIC microscope (63 x/1.4 aperture, 10 × ocular). Images were acquired using the Motic Images Plus 3.0.