Generating High-Titer HIV-1 Stocks

HIV-1 stocks were produced by calcium-phosphate transfection (Promega, Madison, WI) of 293T cells, using 20 μg of pNL4-3 [21 (link)]. Virus preparations were filtered through a 0.2-μm polyvinylidene fluoride filter and quantified by p24 ELISA. Virus was then amplified on CD8-depleted peripheral blood mononuclear cells (PBMCs). Briefly, CD8-depleted PBMCs were isolated from healthy donors, using the EasySep Human CD8 Positive Selection Kit (Stemcell), and were activated in RP10 medium with PHA (5 μg/mL) and IL-2 (30 U/mL) for 3 days. Activated CD8-depleted PBMCs were then infected with HIV_NL4.3 stocks, and 2 feeder cycles of activated, CD8-depleted PBMCs were added 7 and 10 days after infection. Culture supernatant was harvested 14 days after infection, and HIV_NL4.3 virus preparations were filtered and quantified by p24 ELISA.

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Ranganath N., Sandstrom T.S., Burke Schinkel S.C., Côté S.C, & Angel J.B. (2017). The Oncolytic Virus MG1 Targets and Eliminates Cells Latently Infected With HIV-1: Implications for an HIV Cure. The Journal of Infectious Diseases, 217(5), 721-730.

Publication 2017

EasySep Human CD8 Positive Selection Kit

293t cells Calcium phosphate Donors Elisa Hiv 1 Human Infection Peripheral blood mononuclear cells Polyvinylidene fluoride Promega Rp10 Stemcell Transfection Virus

Corresponding Organization : Ottawa Hospital

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Variable analysis

independent variables

Calcium-phosphate transfection of 293T cells with 20 μg of pNL4-3 plasmid

dependent variables

Virus production and quantification by p24 ELISA
Virus amplification on CD8-depleted peripheral blood mononuclear cells (PBMCs)

control variables

Use of 293T cells for transfection
Filtration of virus preparations through a 0.2-μm polyvinylidene fluoride filter
Activation of CD8-depleted PBMCs with PHA (5 μg/mL) and IL-2 (30 U/mL) for 3 days
Addition of 2 feeder cycles of activated, CD8-depleted PBMCs at 7 and 10 days after infection

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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