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AtT20 pituitary corticotroph tumor cells were obtained from ATCC (Manassas, VA, USA). Cells were cultured in DMEM with 10% FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin in T75 culture flasks at 37°C under a humidified 5% CO2 atmosphere. AtT20 cells were subsequently cultured in six-well plates at a density of 1.5×105 cells/well for 3 days prior to each experiment, and culture medium was exchanged with fresh medium every 48 hours. Exogenous factors within the FBS were minimized 1 day prior to each experiment by washing and serum-starving the AtT20 cells overnight with DMEM supplemented with 0.2% bovine serum albumin. Total cellular RNA or protein was collected at the conclusion of each experiment and then stored at −80°C until use. Both PTTG1- and GADD45β-specific siRNA fragments and control siRNAs were designed. All siRNA fragments were purchased from Qiagen. HiPerfect transfection reagent (Qiagen) was used to transfect AtT20 cells with siRNA fragments following the manufacturer’s protocol. Target mRNA levels in samples were determined from cells that were seeded in 12-well plates at a density of 12×104 cells/well; cultures were incubated for 48 hours in 1 mL of culture medium containing control siRNA (siControl) or the experimental siRNAs, that is, the PTTG1-specific siRNA (siPTTG1; Mm_Pttg1_7) or the GADD45β-specific siRNA (siGADD45β; Mm_GADD45β_4). Cell proliferation was measured from cells cultured in 200 µL of culture medium containing siRNA fragments in 96-well plates (1.5×104 cells/well density); the medium was changed after 24 hours of incubation. Cell viability was measured at 48 hours post-transfection using the Cell Counting Kit 8. |
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| AtT20 pituitary corticotroph tumor cells were obtained from ATCC (Manassas, VA, USA). Cells were cultured in DMEM with 10% FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin in T75 culture flasks at 37°C under a humidified 5% CO2 atmosphere. AtT20 cells were subsequently cultured in six-well plates at a density of 1.5×105 cells/well for 3 days prior to each experiment, and culture medium was exchanged with fresh medium every 48 hours. Exogenous factors within the FBS were minimized 1 day prior to each experiment by washing and serum-starving the AtT20 cells overnight with DMEM supplemented with 0.2% bovine serum albumin. Total cellular RNA or protein was collected at the conclusion of each experiment and then stored at −80°C until use. Both PTTG1- and GADD45β-specific siRNA fragments and control siRNAs were designed. All siRNA fragments were purchased from Qiagen. HiPerfect transfection reagent (Qiagen) was used to transfect AtT20 cells with siRNA fragments following the manufacturer’s protocol. Target mRNA levels in samples were determined from cells that were seeded in 12-well plates at a density of 12×104 cells/well; cultures were incubated for 48 hours in 1 mL of culture medium containing control siRNA (siControl) or the experimental siRNAs, that is, the PTTG1-specific siRNA (siPTTG1; Mm_Pttg1_7) or the GADD45β-specific siRNA (siGADD45β; Mm_GADD45β_4). Cell proliferation was measured from cells cultured in 200 µL of culture medium containing siRNA fragments in 96-well plates (1.5×104 cells/well density); the medium was changed after 24 hours of incubation. Cell viability was measured at 48 hours post-transfection using the Cell Counting Kit 8. |
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