Phrodo se

DCPIB (Tocris), NPPB (Tocris), FFA (Sigma), Cytochalasin D (Sigma) and Calcein‐AM (Biolegend) were obtained as powders dissolved in DMSO to ×200 concentrated stocks and stored at −20°C. pHrodo‐SE (ThermoFisher) was dissolved in DMSO to 10 mg/ml and stored in aliquots at −80°C. Human Aβ_1‐42 (Eurogentec) was dissolved in hexafluoro‐2‐propanol, dried into films under a rotary evaporator (Eppendorf) and stored at −80°C. Tomato lectin conjugated to DyLight 594 (Vector Biolabs) was washed twice through a centrifugal filter (10 kDa MWCO) to remove sodium azide, sterile filtered and stored as a 1 mg/ml stock at 4°C.

To assess the influence of serum components in ApoCell-phagocytosis, apoptotic cells were pre-treated (for 15-min at 37°C) with DMEM culture medium alone, with whole serum or with purified IgG preparations (50 µg/ml) derived from autoimmune patients or HBD. Apoptotic cells were washed and incubated (1×10⁶ cells) with preparations of isolated peripheral blood mononuclear cells (1×10⁶ cells) or MDM (1×10⁵ cells) derived from a healthy donor, and the samples were analyzed for ApoCell-phagocytosis, as above. In separate experiments, prior the addition to mixtures, serum samples from HBD were heat-inactivated (56°C, 30-min) to destroy complement. The rates of ApoCell-phagocytosis by healthy MDM of apoptotic cells pretreated with purified IgG preparations (50 µg/ml) derived from either autoimmune patients or HBD were also comparatively investigated by two-color flow cytometry analyses of healthy MDM stained with CD14-PE (BD-Pharmingen) and CFSE-labelled IgG-pretreated apoptotic cells, as well as by single-color flow cytometry using apoptotic cells labelled with the pH-sensitive fluorescent dye pHrodo succinimidyl ester (pHrodo-SE, Life Technologies, 20 ng/ml at room temperature for 30-min), which allows the actual detection of ingested apoptotic cells owing to increased light emission in the acidic environment of the phagosomes of phagocytes [27] (link).