Glut1 siRNA and shRNA Plasmids (h)

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	Human bladder cancer cell lines UM-UC-3 and HT-1376 derived from high-grade transitional cell carcinoma (from the American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC) supplemented with 10% (v/v) of fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B (Sigma). GLUT1 was depleted in human bladder cancer cells using a pool of three target-specific 20–25 nt siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). HT-1376 bladder cancer cells were transfected in 6- or 96-well culture plates, at 60–80% confluence, with GLUT1. 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. 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.

Protocol tips

Human bladder cancer cell lines UM-UC-3 and HT-1376 derived from high-grade transitional cell carcinoma (from the American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC) supplemented with 10% (v/v) of fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B (Sigma). GLUT1 was depleted in human bladder cancer cells using a pool of three target-specific 20–25 nt siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). HT-1376 bladder cancer cells were transfected in 6- or 96-well culture plates, at 60–80% confluence, with GLUT1. 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. 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.

Publication protocol

Human bladder cancer cell lines UM-UC-3 and HT-1376 derived from high-grade transitional cell carcinoma (from the American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC) supplemented with 10% (v/v) of fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B (Sigma). GLUT1 was depleted in human bladder cancer cells using a pool of three target-specific 20–25 nt siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). HT-1376 bladder cancer cells were transfected in 6- or 96-well culture plates, at 60–80% confluence, with GLUT1. 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. 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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