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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
| Cells were seeded at 2×105 cells/well in 6-well plates (Corning Incorporated, Corning, NY, USA) and grown to 60% confluence on the day of transfection. |
The media was removed from the plate wells and replaced with 2 ml complete medium containing Polybrene (cat. no., sc-134220; Santa Cruz Biotechnology, Inc.) at a final concentration of 5 µg/ml. Cells were transfected with control (scramble, 20 µl) or CD74 shRNA lentiviral particles (20 µl) diluted in media according to the manufacturer's protocol in 48 h. |
Infected cells were selected with puromycin (5 µg/ml; cat. no., 11811-023; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). A total of 72 h after transfection, transfected cells were verified by RT-PCR and western blotting analysis, comparing untreated and scramble cells. |
| Upstream tips |
| Cells were seeded at 2×105 cells/well in 6-well plates (Corning Incorporated, Corning, NY, USA) and grown to 60% confluence on the day of transfection. |
| Protocol tips |
| The media was removed from the plate wells and replaced with 2 ml complete medium containing Polybrene (cat. no., sc-134220; Santa Cruz Biotechnology, Inc.) at a final concentration of 5 µg/ml. Cells were transfected with control (scramble, 20 µl) or CD74 shRNA lentiviral particles (20 µl) diluted in media according to the manufacturer's protocol in 48 h. |
| Downstream tips |
| Infected cells were selected with puromycin (5 µg/ml; cat. no., 11811-023; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). A total of 72 h after transfection, transfected cells were verified by RT-PCR and western blotting analysis, comparing untreated and scramble cells. |
| Upstream tips |
Protocol tips |
Downstream tips |
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cancer cells were transfected in 6- or 96-well culture plates, at 60–80% confluence, with galectin-1 and GLUT1, respectively. Cells were also transfected with a scrambled siRNA in parallel as controls.
For each transfection, cells were treated for 5 h with 2.4 µM of siRNA in transfection medium (Santa Cruz) containing 0.5 µL/cm2 of transfection reagent (Santa Cruz). After incubation, complete media was added and the cells were incubated for 24 or 48 h. Galectin-1 or GLUT1 downregulation was evaluated 24 h or 48 h post-transfection by Western blotting. The uptake and PDT experiments were performed 24 h or 48 h post-transfection with GLUT1 hsiRNA or galectin-1 hsiRNA, respectively. |
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| Protocol tips |
cancer cells were transfected in 6- or 96-well culture plates, at 60–80% confluence, with galectin-1 and GLUT1, respectively. Cells were also transfected with a scrambled siRNA in parallel as controls.
For each transfection, cells were treated for 5 h with 2.4 µM of siRNA in transfection medium (Santa Cruz) containing 0.5 µL/cm2 of transfection reagent (Santa Cruz). After incubation, complete media was added and the cells were incubated for 24 or 48 h. Galectin-1 or GLUT1 downregulation was evaluated 24 h or 48 h post-transfection by Western blotting. The uptake and PDT experiments were performed 24 h or 48 h post-transfection with GLUT1 hsiRNA or galectin-1 hsiRNA, respectively. |
| Upstream tips |
Protocol tips |
Downstream tips |
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Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to perform all cell transfections with 10 nM vectors or 50 nM siRNA and using aforementioned cell lines at a density of 1×106 cells in 2 ml cell suspension. Treatment with Lipofectamine® 2000 reagent alone was used as the C group and transfection with empty vectors or NC siRNA was used as the NC group. Cells were used for subsequent experiments when LINC01638 and ROCK2 overexpression rates were >200% and ROCK2 knockdown rate was <50%, only. The time interval between transfection and subsequent experimentation was 24 h. |
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| Protocol tips |
| Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to perform all cell transfections with 10 nM vectors or 50 nM siRNA and using aforementioned cell lines at a density of 1×106 cells in 2 ml cell suspension. Treatment with Lipofectamine® 2000 reagent alone was used as the C group and transfection with empty vectors or NC siRNA was used as the NC group. Cells were used for subsequent experiments when LINC01638 and ROCK2 overexpression rates were >200% and ROCK2 knockdown rate was <50%, only. The time interval between transfection and subsequent experimentation was 24 h. |
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