To generate a B16F10 cell line stably expressing GFP, B16F10 cells at 50% confluence were infected with the second-generation GFP-IRES-GFP, CBG, or FLuc lentivirus. GFP-IRES-GFP– and CBG-infected B16F10 cells were selected for GFP expression by fluorescence-activated cell sorting. FLuc-infected B16F10 cells were selected by bioluminescence imaging. After 2 weeks, bioluminescence imaging of isolated cell colonies using phenol-free DMEM with 10% FBS and d-luciferin (150 μg/ml) was acquired to assess reporter gene expression. Bioluminescent colonies were harvested and expanded. Cells were transduced at a multiplicity of infection (MOI) of 10.

To generate B16F10 GFP-IRES-GFP cell lines stably expressing pκB5→IκBα-FLuc or pκB5→FLuc, cells at 95% confluence were cotransfected with 10 μg of either pκB5→IκBα-FLuc or pκB5→FLuc plasmid and 3 μg of pLVX-IRES-Puro plasmid DNA using FuGENE 6 transfection reagent (Promega, Madison, WI, USA) in a six-well plate. At 24 hours after transfection, media was replaced with fresh cell media, and at 48 hours after transfection, cells were trypsinized and plated at a variety of densities (1:2, 1:5, and 1:10) into media containing puromycin (2 μg/ml) to select for stable transformants. After 2 weeks, bioluminescence imaging of isolated cell colonies using phenol-free DMEM with 10% FBS and d-luciferin (150 μg/ml) was performed to assess reporter gene expression. Bioluminescent colonies were harvested and expanded. Cells were continuously cultured in the presence of puromycin (2 μg/ml) to maintain expression of the reporter plasmid.

To generate a B16F10 cell line stably expressing dendra2, cells at 95% confluence were cotransfected with 10 μg of pDendra2-N plasmid using FuGENE 6 in a six-well plate. At 24 hours after transfection, media was replaced with fresh cell media, and at 48 hours after transfection, cells were trypsinized and plated in media containing G418 (1.5 mg/ml) to select for stable transformants. Flow cytometry was used to isolate dendra2-positive cells; cells were sorted twice for dendra2 fluorescence (λex = 409 nm, λem = 507 nm).

To generate a B16F10 dendra2 cell stably expressing NF-κB→FLuc, B16F10 dendra2 cells at 50% confluence were transduced with the NF-κB→FLuc lentivirus and selected using puromycin (2 μg/ml). Cells were transduced at an MOI of 10. All cell lines tested negative for mycoplasma.

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