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Found 1 matching solution for this experiment
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PC3 (bone metastatic PCa cell line, ATCC®-CRL-1435), was purchased from ATCC. PC3 cells were cultured in RPMI-1640 medium containing 10% FBS, 2.5 mM of L-glutamine and 1% penicillin/streptomycin. Both cell lines were cultured at 37 °C in a humidified tissue culture incubator containing 5% CO2. Cells were maintained and propagated at 33 °C and 5% CO2 except during co-culture experiments which were conducted at 37 °C. All cell lines were periodically checked for mycoplasma using MycoAlert™ Mycoplasma Detection Kit (Lonza). Authentication of cell lines was performed by STR DNA profiling analysis conducted by the Molecular Resources Facility at Rutgers University. Cell populations were frozen after 3 passages from the time of initial receipt and growth and were discarded after 30 passages.HSPA5 (GRP78) targeting siRNAs were purchased from Ambion (Carlsbad, USA). Two different siRNAs; Silencer® Select Pre-designed siRNA s6979 (5’ UUC UGG ACG GGC UUC AUA Gtt 3′) and s6980 (5’ UCU AGU AUC AAU GCG CUC Ctt 3′) targeting exons 6 and 8, respectively, were tested. For control, the Silencer® select negative control No. 2 siRNA was used (Ambion). siRNA transfections were performed using a modified reverse transfection technique [32] using a cocktail containing equimolar quantities of each GRP78 siRNA to maximize silencing potential. The GRP78 siRNA cocktail (or siRNA control) was diluted in Opti-MEM reduced serum medium and incubated with the TransIT-X2 dynamic delivery system (Mirus Bio) according to the manufacturer’s protocol. The siRNA-TransIT-X2 complexes were added to wells of either a 6- or 24- well plate upon which PC3 cells seeded in complete growth medium at a cell density of 7.5-9 × 105 cells/well (6 well plate) or 0.75-1 × 105 cells/well (24 well plate). GRP78 siRNA cocktail or control siRNA were used at a final concentration of 50 nM for PC3 cell line. |
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| Protocol tips |
| PC3 (bone metastatic PCa cell line, ATCC®-CRL-1435), was purchased from ATCC. PC3 cells were cultured in RPMI-1640 medium containing 10% FBS, 2.5 mM of L-glutamine and 1% penicillin/streptomycin. Both cell lines were cultured at 37 °C in a humidified tissue culture incubator containing 5% CO2. Cells were maintained and propagated at 33 °C and 5% CO2 except during co-culture experiments which were conducted at 37 °C. All cell lines were periodically checked for mycoplasma using MycoAlert™ Mycoplasma Detection Kit (Lonza). Authentication of cell lines was performed by STR DNA profiling analysis conducted by the Molecular Resources Facility at Rutgers University. Cell populations were frozen after 3 passages from the time of initial receipt and growth and were discarded after 30 passages.HSPA5 (GRP78) targeting siRNAs were purchased from Ambion (Carlsbad, USA). Two different siRNAs; Silencer® Select Pre-designed siRNA s6979 (5’ UUC UGG ACG GGC UUC AUA Gtt 3′) and s6980 (5’ UCU AGU AUC AAU GCG CUC Ctt 3′) targeting exons 6 and 8, respectively, were tested. For control, the Silencer® select negative control No. 2 siRNA was used (Ambion). siRNA transfections were performed using a modified reverse transfection technique [32] using a cocktail containing equimolar quantities of each GRP78 siRNA to maximize silencing potential. The GRP78 siRNA cocktail (or siRNA control) was diluted in Opti-MEM reduced serum medium and incubated with the TransIT-X2 dynamic delivery system (Mirus Bio) according to the manufacturer’s protocol. The siRNA-TransIT-X2 complexes were added to wells of either a 6- or 24- well plate upon which PC3 cells seeded in complete growth medium at a cell density of 7.5-9 × 105 cells/well (6 well plate) or 0.75-1 × 105 cells/well (24 well plate). GRP78 siRNA cocktail or control siRNA were used at a final concentration of 50 nM for PC3 cell line. |
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