Erythrocyte dehydration is associated with adhesion molecule activation. (A) Intracellular K⁺ content in aged, sickle cell, and stored erythrocytes was measured using ion-specific electrodes (n = 4-8; 1-way ANOVA). (B) Erythrocyte deformability was determined using the automated rheologic cell analyzer (ARCA). A shear stress of 30 dyne/cm² was applied to determine deformability (ratio of length (A) to width (B) of the cell (n = 3; 1-way ANOVA). (C) Biotinylated Maackia amurensis (MA) lectin was used to quantify α2,3-linked sialic acid (α2,3-SIA) on aged, stored (in SAGM medium), and sickle erythrocytes; data were normalized (Norm.) to untreated controls (n = 3; 1-way ANOVA). (D) A total of 10⁷ control, aged, stored, and sickle erythrocytes were flowed over laminin-α5 at 0.2 dynes/cm² and adhesion frequency was assessed by microscopy (n = 3-7; 1-way ANOVA). (E) The same flow experiment was performed on HA. Here, rolling frequency was quantified instead of adhesion (n = 3-7; 1-way ANOVA). (F) Intracellular K⁺ content in control and valinomycin-treated cells was measured using ion-specific electrodes over 60 minutes. (G) ARCA was performed to assess deformability of erythrocytes treated for 30 minutes with valinomycin (n = 3). (H) Biotinylated M amurensis lectin was used to quantify α2,3-linked sialic acid on erythrocytes exposed for 30 minutes to valinomycin; data were normalized to untreated controls (n = 3-4, 1-way ANOVA). (I) A total of 10⁷ control and valinomycin-treated erythrocytes were flowed over laminin-α5 and HA at 0.2 dynes/cm² and adhesion frequency was assessed by microscopy. Erythrocytes were treated with valinomycin for 30 minutes because this results in levels of intracellular potassium similar to those in aged, sickle, and stored erythrocytes (panel A) (n = 4-9; Student t test). (J) Flow cytometric quantification of α2,3-linked sialic acid on old, stored, and valinomycin-treated erythrocytes (n = 3-4; 1-way ANOVA). (K) Erythrocytes were stored for up to 4 weeks at 4°C during which adhesion frequency to laminin-α5 and rolling frequency on HA was assessed. Significance compared with T = 0 is indicated (1-way ANOVA). Error bars indicate standard deviation unless otherwise noted. *P < .05; **P < .01; ***P < .001; ****P < .0001. Hb, hemoglobin; MFI, mean fluorescence intensity; ns, not significant; RBC, red blood cell.