Ph rodamine

To assess the capacity of myeloid cells to ingest myelin, we first incubated the purified human myelin with a pH-sensitive dye (pH-Rodamine; Invitrogen) for 1 h in PBS (pH = 8). Fluorecently-labeled myelin was then added at a final concentration of 20 μg/mL to microglia or macrophage that were pre-exposed to distinct B-cell supernatants for two days. Following 1 h of incubation at 37 °C, epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) was used to visualize and quantify myelin phagocytosis by the microglia and macrophages, and flow cytometry (FACS Fortessa, BD Bioscience) was used to quantify internalized myelin. Macrophage myelin phagocytosis assays were performed to directly compare the impact of pro-inflammatory versus IL-10 expressing B cell supernatants derived from both RRMS patients and healthy controls (HC) on responses of the same HC donor macrophage.

Endotoxin-free human myelin was prepared as previously described.^{14 (link)} To evaluate myelin phagocytosis, MDMs were plated in 12-well plates.^{5 (link)} Myelin was conjugated to a pH-sensitive dye (pH-Rodamine; Invitrogen, Carlsbad, CA) for 1 hour in phosphate-buffered saline (PBS) (pH 8) and added to the culture wells (20 μg/mL) for 1 hour; cells were washed, detached, pelleted, and passed through a flow cytometer (FACS Fortessa; BD Biosciences, San Jose, CA) to quantify the percentage of myeloid cells containing internalized fluorescent myelin. A similar protocol was used to study phagocytosis of red blood cells (RBCs) acquired from Ficoll-Paque density gradient centrifugation and diluted in PBS at a ratio of 1:2,500. RBCs were then incubated with opsonizing antibody (20 μg/mL: Cederlane) and pH-rhodamine for 30 minutes at room temperature. MDMs were exposed to opsonized RBCs for 1 hour at a 4:1 (RBC:MDM) ratio, and internalized RBCs were quantified as an increase in mean fluorescent intensity in the PE channel.