Ngf β

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.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of HIV seronegative donors by density gradient centrifugation over Ficoll–Paque Plus (GE Healthcare). CD14⁺ monocytes cells were isolated from PBMCs using human CD14 microbeads beads (Miltenyi Biotech).
Monocyte-derived macrophages (MDM) were generated in vitro by culturing CD14⁺ monocytes in RPMI 1640 (Gibco) supplemented with 10 % (v/v) charcoal dextran-treated, heat-inactivated FBS (Gemini Bio-Products), 1 % antibiotics (10,000 units/ml penicillin, 10,000 μg/ml streptomycin, Gibco), and 10 ng/ml macrophage colony-stimulating factor (Peprotech) for 10 days at 37 °C, 5 % CO₂. MMG cells were generated from CD14⁺ monocytes using methods modified from those originally described by Etemand et al. (Etemad et al. 2012 (link)). CD14⁺ monocytes isolated from healthy human donor blood were cultured in RPMI-1640 GlutaMax (Gibco) supplemented with 1 % antibiotics (10,000 units/ml penicillin 10,000 μg/ml streptomycin, Gibco) and mixture of human recombinant cytokines including M-CSF (10 ng/ml; Peprotech), beta-nerve growth factor (NGF-β 10 ng/ml; R&D Systems), and CCL2 (100 ng/ml; Peprotech). Every third day, cells were supplemented with fresh media containing M-CSF, GM-CSF, NGF-β, and CCL2. Cells were cultured for 10 days. Most of the characterization experiments were performed on day 10.