Protocol detail

Lentiviral Transduction and Cell Sorting

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For virus generation, Lenti-X HEK 293T cells (Takara Bio) were transfected with lentiviral packaging plasmids and transgenic constructs. Virus was harvested and clarified through a 0.45-μm filter after 3 d. Lentiviral titering was optimized to result in <50% transduction to generate cells with MOI < 1, and cells were infected via centrifugation at 150g for 1 h in 8 μg/ml polybrene (EMD Millipore). After 3 d, transduced cells were selected via FACS using an 85–100 μm-diameter nozzle. ATF4 reporter cell lines were generated and sorted following stress induction as previously described (Guo et al, 2020 (link)).

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Park J., Desai H., Liboy-Lugo J.M., Gu S., Jowhar Z., Xu A, & Floor S.N. (2024). IGHMBP2 deletion suppresses translation and activates the integrated stress response. Life Science Alliance, 7(8), e202302554.

Publication 2024

Lenti-X HEK 293T cells polybrene

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Variable analysis

independent variables

Lentiviral packaging plasmids and transgenic constructs
Lentiviral titering to result in <50% transduction to generate cells with MOI < 1
Centrifugation at 150g for 1 h in 8 μg/ml polybrene
Stress induction to generate ATF4 reporter cell lines

dependent variables

Lentiviral titer
Transduction efficiency
ATF4 reporter activity

control variables

Lenti-X HEK 293T cells
Virus harvesting and clarification through a 0.45-μm filter after 3 days
Cell selection via FACS using an 85–100 μm-diameter nozzle

controls

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

Annotations

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