Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of HIV seronegative donors using density gradient centrifugation over Ficoll–Paque Plus (GE Healthcare Cat# 114402). Primary human monocytes were isolated from PBMCs using monocyte isolation kit II, human (Miltenyi Biotech Cat# 130–091-103). Human microglia cells (HMG) were differentiated from primary human monocytes using methods as described previously (Etemad, Zamin, Ruitenberg, & Filgueira, 2012 (link); Rawat & Spector, 2017 (link)). Briefly, monocytes were seeded at 2.5×105 cells/well in a 48-well plate in serum free RPMI Glutamax medium (Gibco Cat# 61870036) supplemented with MCSF (10ng/ml; Peprotech Cat# 300–25), MCP1 (100ng/ml; Peprotech Cat# 300–04), GMCSF (10ng/ml; Peprotech Cat# 300–03) and NGF-β (10ng/ml; R&D Systems Cat# 256-GF) with media changed every 3 days.
For autophagy induction in HMG cells, culture medium was supplemented with trehalose (Sigma, Cat# T0167) at 100mM concentration. The lysosomal degradation inhibitor bafilomycin A1 (Cell Signaling Cat# 54645) was prepared in dimethyl sulfoxide (DMSO) and added to the medium at a concentration of 10nM to measure the autophagic flux. DMSO was added to the medium of untreated cells as vehicle control.
For autophagy induction in HMG cells, culture medium was supplemented with trehalose (Sigma, Cat# T0167) at 100mM concentration. The lysosomal degradation inhibitor bafilomycin A1 (Cell Signaling Cat# 54645) was prepared in dimethyl sulfoxide (DMSO) and added to the medium at a concentration of 10nM to measure the autophagic flux. DMSO was added to the medium of untreated cells as vehicle control.
