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Extra resources for Human Retrovirus Protocols: Virology and Molecular Biology (Methods in Molecular Biology 304)
The cells are centrifuged at 500g for 7 min and resuspended in 5 mL supplemented RPMI medium. The CD8+ T-cells (approx 10% of total PBMC) are depleted using anti-CD8 beads according to the manufacturer’s instructions. First, the anti-CD8 beads are washed by transferring the required number of beads (100 µL of beads per 1 × 107 PBMC) into a 15-mL tube and resuspending them in 1 mL of supplemented RPMI medium. The tube is placed on a magnetic particle concentrator for 3 min and then the medium is gently removed using a sterile transfer pipet.
We have also found that LPS stimulation of patient monocytes, or stimulation with other agents including M-CSF or tumor necrosis factor (TNF)-_ prior to co-culture with CD8depleted PHA-activated PBMC, may increase the amount of virus isolated, or decrease the number of monocytes required for successful virus isolation. However, successful virus isolation can be achieved in approx 70% of cases without any prior stimulation step (2). 8. A common concern during the monocyte-PBMC co-culture is the relatively high density at which the cells are cultured.
NEK050B001KT). 8. 5 kit (Roche Diagnostics, cat. no. 1118390) (see Note 4). 3. 1. Isolation of Peripheral Blood Monocytes From PBMC of HIV-1-Infected Individuals The isolation of peripheral blood monocytes requires (1) the preparation of the PBMC fraction from peripheral blood, (2) the isolation of monocytes from the total PBMC, and (3) testing the purity of the isolated monocytes by flow cytometry. 1. Preparation of PBMC Fraction From Peripheral Blood Peripheral blood mononuclear cells are isolated from the patient blood by first diluting the blood 1:2 with PBS-CMF and gently overlaying 30 mL of the diluted blood onto 15 mL of Ficoll-Hypaque in two 50-mL conical centrifuge tubes.