In vivo, OLs do not exist as an isolated population. Rather, they formed a complex network with all the other major cells types in the CNS. Signaling between OLs and neurons, astrocytes and microglia have been reported plenty (Bastmeyer et al., 1991; Schwab and Schnell, 1991). To further assess whether the injury mechanism we observed in vitro faithfully reflects the real HIV pathogenesis in vivo, we used two models, a co-culture model where OLs are cultured on top of a mixed glial layer and a brain aggregate model where dissociated progenitors acquired from mice or human fatal tissues were supplied with serum and allowed to aggregate spontaneously.
Co-culture of neurons and mixed glia have been used to study the synergistic toxic effects of HIV-1 Tat and morphine on neurons (Zou et al., 2011b). In this model, a confluent layer of mixed primary glial …show more content…
In short, leukopak were diluted with PBS-EDTA in a 1:1 ratio before loaded on top of the LSM layer (30 ml diluted blood per 12.5 ml LSM) in a 50 ml tube and centrifuged at 2000 rpm for 30 min at room temperature. Cells were then collected and re-suspended in PBS-EDTA (Miltenyi Biotec Inc. San Diego, CA), and treated with ACK lysis buffer (Life technologies) for 5 min at room temperature before cultured in T150 flash (Corning) in 20 ml HMDM medium [RPMI1640 medium supplied with L-Glutamine (1%), fetal bovine serum (10%) and penicillin/streptomycin (1%)] overnight. At the next day, cells were collected and re-plated in T75 flasks in a density of 40 million cells per 20 ml HMDM per flask. Phytohemagglutinin (PHA-M) were added in the medium to a final concentration of 5 μg/mL to activate the PBMCs. After activation, 1 ng HIV-1 p24/ml HIV-1Ba-L virus, measured by HIV-1 p24 ELISA (ABL inc., Rockville, MD), will be added to the culture medium to infect the PBMCs for 3
Co-culture of neurons and mixed glia have been used to study the synergistic toxic effects of HIV-1 Tat and morphine on neurons (Zou et al., 2011b). In this model, a confluent layer of mixed primary glial …show more content…
In short, leukopak were diluted with PBS-EDTA in a 1:1 ratio before loaded on top of the LSM layer (30 ml diluted blood per 12.5 ml LSM) in a 50 ml tube and centrifuged at 2000 rpm for 30 min at room temperature. Cells were then collected and re-suspended in PBS-EDTA (Miltenyi Biotec Inc. San Diego, CA), and treated with ACK lysis buffer (Life technologies) for 5 min at room temperature before cultured in T150 flash (Corning) in 20 ml HMDM medium [RPMI1640 medium supplied with L-Glutamine (1%), fetal bovine serum (10%) and penicillin/streptomycin (1%)] overnight. At the next day, cells were collected and re-plated in T75 flasks in a density of 40 million cells per 20 ml HMDM per flask. Phytohemagglutinin (PHA-M) were added in the medium to a final concentration of 5 μg/mL to activate the PBMCs. After activation, 1 ng HIV-1 p24/ml HIV-1Ba-L virus, measured by HIV-1 p24 ELISA (ABL inc., Rockville, MD), will be added to the culture medium to infect the PBMCs for 3