Journal: PLoS ONE

Article Title: Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function

doi: 10.1371/journal.pone.0141206

Figure Lengend Snippet: (A) RBCs from septic patients show increased presence of C4d fragments, but not C3d. RBCs isolated from patient samples were incubated with anti-C3d or anti-C4d and analyzed by flow cytometry. (B) RBCs from septic patients do not show uniform increase in band 3 phosphorylation. RBCs from septic and control patients were incubated with EMA, washed twice, and band 3 phosphorylation was measured by flow cytometry. (C) ROS levels increased in septic RBCs. RBCs were incubated with DHR 123, washed twice, and measured for intracellular ROS by flow cytometry. (D) RBCs from septic patients release less ATP than control RBCs. RBCs from septic and control patients were isolated and ATP concentration was measured as described in Methods. (E) Sepsis-induced decrease in RBC lipid mobility depends on ROS. RBCs from control and septic patients were incubated with DiO lipid dye in the presence or absence of GSH-ME (1 mM) prior to running FRAP. (F) Sepsis-dependent loss of RBC membrane deformability depends on ROS. RBCs from control and septic patients were washed and loaded into a 2D microfluidic device in the presence or absence of GSH-ME (1 mM, RT x 15 min). Sequential images (3 frames/sec) were recorded of each cell’s passage through the channel.

Article Snippet: Antibodies were obtained as follows—LEAF purified mouse IgG 1 (401404); LEAF purified mouse IgM (401604, Biolegend, San Diego, CA, USA); mouse anti-phosphotyrosine mAb (Life Technologies, Carlsbad, CA, USA); mouse monoclonal anti-beta-actin Ab (A2228), mouse monoclonal anti-glycophorin A IgG (clone R10, Lot B0607, sc-53905); mouse monoclonal anti-GPA IgM (clone E4, G7900, Sigma-Aldrich, St Louis, MO, USA); anti-CR1 mAb 1F11 (gift of Henry Marsh, Celldex Therapeutics, Needham, MA, USA); mouse monoclonal anti-CFTR (CF3) (ab2784, both Abcam, Cambridge, MA, USA); AffiniPure goat anti-mouse IgG (115-005-003); ChromPure rabbit IgG (011-000-003); ChromPure mouse IgG (015-000-003, Jackson ImmunoResearch, West Grove, PA, USA); HRP conjugated goat anti-mouse antibody (Millipore, Billerica, MA, USA); mouse monoclonal anti-human C3d (A207), mouse monoclonal anti-human C4d (A213, Quidel, San Diego, CA, USA).

Techniques: Isolation, Incubation, Flow Cytometry, Concentration Assay