We generated stable HeLa CCL2 cells expressing YFP-CASP4(C258S) (31 (link)) by lentiviral transduction. Lentivirus was produced in HEK293T by transient transfection with the lentiviral backbone pMX-CMV-YFP CASP4(C258S) and the three packaging plasmids pVSV-G (#138479, Addgene), pAdvantage (E1711, Promega), and pGag-Pol (#14887, Addgene). DNA, mixed with FuGENE 6 Transfection Reagent (Promega) in Opti-MEM (Gibco), was incubated at RT for 15 min, then added dropwise into HEK293T cells. After 48h, the supernatant containing the lentivirus was collected, filtered using a 0.45 μm membrane filter, and supplemented with polybrene (Sigma) at 8 μg/mL. Lentivirus was then transduced into HeLa CCL2 cells by centrifugation of the HEK293T supernatant for 2 h at 1000 × g at 30°C. Two days later, HeLa CCL2 containing YFP-CASP4(C258S) were selected and maintained with 2 μg/mL of puromycin.
Protocol detail
Generating stable HeLa CCL2 cells expressing YFP-CASP4
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Backbone
Casp4
Ccl2
Cells
Centrifugation
Fugene 6
Hela
Lentivirus
Membrane
Pgag
Plasmids
Polybrene
Promega
Puromycin
Transfection
Transient
Corresponding Organization : Broad Institute
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- Lentiviral transduction of HeLa CCL2 cells with YFP-CASP4(C258S)
- Generation of stable HeLa CCL2 cells expressing YFP-CASP4(C258S)
- Transfection of HEK293T cells with lentiviral backbone pMX-CMV-YFP CASP4(C258S) and packaging plasmids pVSV-G, pAdvantage, and pGag-Pol
- Incubation of DNA-FuGENE 6 mixture in Opti-MEM for 15 minutes before adding to HEK293T cells
- Collection of lentivirus-containing supernatant from HEK293T cells after 48 hours
- Filtration of lentivirus-containing supernatant using a 0.45 μm membrane filter
- Supplementation of lentivirus-containing supernatant with polybrene at 8 μg/mL
- Centrifugation of lentivirus-containing supernatant for 2 hours at 1000 × g and 30°C to transduce HeLa CCL2 cells
- Selection and maintenance of HeLa CCL2 cells containing YFP-CASP4(C258S) with 2 μg/mL of puromycin
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