Journal: Frontiers in Immunology

Article Title: Extracellular Vesicles From Stored Red Blood Cells Convey Heme and Induce Spic Expression on Human Monocytes

doi: 10.3389/fimmu.2022.833286

Figure Lengend Snippet: Characterization of RBC-EVs and their effects in monocytes. EVs were isolated from fresh and 21 day-stored-blood by centrifugation at 20,000 x g (A) Heme and (B) hemoglobin within EVs were measured using a plate reader at 398 nm and 540. (C) Oxidized hemoglobin (metHb) was measured at 541, 576 and 630 nm by the following equation: [metHb] = 185.77 x OD541+ 171.88 X OD576 +387.58 X OD630. (D) Isolated monocytes were challenged with 20% (v/v) EVs (fresh or stored) for 30 min, washed and lysed with RIPA buffer on ice, and captured heme was measured at 398 nm within monocytes. (E) Monocytes were treated for 24h with 20% (v/v) of fresh (RBC 0-EVs) or stored EVs (RBC 21-EVs), and protein extracts were subjected to Western Blotting for quantification of Spic expression. (F) Monocytes were treated for 24 hours with metHb at a concentration of 30 µM of heme, and protein extracts were subjected to Western Blotting to quantify Spic expression. (G) Monocytes were treated for 24 hours with RBC 0-EVs, and RBC 21-EVs previously incubated or not with hemopexin (Hpx; 1 µM for 1 hour), and protein extracts were subjected to Western Blotting for quantification of Spic expression. The results are representative of 3 to 5 different experiments. Data are expressed as mean ± standard error (SEM). *Represents p < 0.05; ***represents p < 0.005.

Article Snippet: Cell lysates (10-20 μg of protein) were fractionated by polyacrylamide 12 or 15% (SDS-PAGE) gels (1 hour at 150V and 50mA, PowerPacTM HC, Bio-Rad, Singapore).

Techniques: Isolation, Centrifugation, Western Blot, Expressing, Concentration Assay, Incubation