third generation lentiviral packaging plasmid mix  (Addgene inc)

Journal: Scientific Reports

Article Title: Functional multiplex reporter assay using tagged Gaussia luciferase

doi: 10.1038/srep01046

Figure Lengend Snippet: (a) Schematic of the lentiviral vector constructs encoding the luciferase reporters. (b) Fluorescence microscopy analysis of a representative U87-FM-Gluc tag -CFP cells (using Gluc Flag reporter) showing mCherry levels (in red; marker for tumor cells) and CFP expression (in blue; marker for transduction efficiency). (c) Immunostaining against the various tags in U87-FM cells expressing different Gluc tag . (d) Gluc activity in U87 cells expressing different Gluc tag with respect to cell proliferation over time and cell number. The dashed line represents Gluc Ctrl activity. (e) Gluc tag immunobinding assay versus total Gluc activity using serial dilutions of the conditioned medium from a 1-day culture of U87-FM cell lines expressing the individual Gluc tag reporters. (f-g) Equal mixture of ten cell lines each expressing a different Gluc tag reporter. (f) Immunobinding assay of Gluc tag from a mixed population. (g) Immunostaining for the ten different tags expression in the mixed population of U87-FM cell culture. (h-i) U87-FM cells expressing all ten different Gluc tag reporters. (h) Immunostaining for the ten different tags in one cell line. The insert panels show staining of control parental cells. Percentages of positively stained cells are indicated. (i) Immunobinding assay of all ten Gluc tag reporters in one cell line after TMZ treatment (black bars) compared to untreated control (white bars). (j) CFP fluorescence microscopy of cells in (h–i) with or without 600 μM TMZ. Size bar (in b,c, g and h) = 200 μm. Size bar (i) = 400 μm. N.d. = not detectable.

Article Snippet: The Gluc tag construct was co-transfected with a third generation lentiviral packaging mix (pMDLg/pRRE, pRSV-Rev and pMD2.G, Addgene) in HEK293T cells using Lipofectamine 2000 (Life Technologies).

Techniques: Plasmid Preparation, Construct, Luciferase, Fluorescence, Microscopy, Marker, Expressing, Transduction, Immunostaining, Activity Assay, Cell Culture, Staining, Control