AChE shRNA Plasmids (h)

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	TF‐1 cells (CRL‐2003; ATCC; Manassas, VA; RRID:CVCL_0559), an erythroleukemic cell line from human blood, were maintained at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mM sodium pyruvate, 4.5 g/L glucose and 2 ng/mL GM‐CSF (PHC2015). Prior to induction of differentiation, cells were kept in medium without GM‐CSF overnight, followed by the addition of EPO (1 U/mL; PHC2054) on the next day. The TF‐1 cell line is not listed in the database of commonly misidentified cell lines (International Cell Line Authentication Committee), and not authenticated by the authors. All culture reagents were from Life Technologies (Life Technologies, Carlsbad, CA, USA). The renewal of medium and EPO (1 U/mL) were done on alternative days, and the cell density was maintained at 4 × 105 cells/mL. Cultured TF‐1 cells were transfected with cDNA constructs with Trans IT X2® Dynamic Delivery System (Mirus BIO, Madison, WI, USA) for AChE shRNA and (Santa Cruz, Dallas, TX, USA). Transfection was performed 1 day after cells were plated. For every 5 × 106 cells in 1 mL medium, DNA: Trans IT X2® were mixed in the ratio of 1 : 2 (3 μg/6 μL) in antibiotic free medium and added to the cells within 20 min.

Protocol tips

TF‐1 cells (CRL‐2003; ATCC; Manassas, VA; RRID:CVCL_0559), an erythroleukemic cell line from human blood, were maintained at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mM sodium pyruvate, 4.5 g/L glucose and 2 ng/mL GM‐CSF (PHC2015). Prior to induction of differentiation, cells were kept in medium without GM‐CSF overnight, followed by the addition of EPO (1 U/mL; PHC2054) on the next day. The TF‐1 cell line is not listed in the database of commonly misidentified cell lines (International Cell Line Authentication Committee), and not authenticated by the authors. All culture reagents were from Life Technologies (Life Technologies, Carlsbad, CA, USA). The renewal of medium and EPO (1 U/mL) were done on alternative days, and the cell density was maintained at 4 × 105 cells/mL. Cultured TF‐1 cells were transfected with cDNA constructs with Trans IT X2® Dynamic Delivery System (Mirus BIO, Madison, WI, USA) for AChE shRNA and (Santa Cruz, Dallas, TX, USA). Transfection was performed 1 day after cells were plated. For every 5 × 106 cells in 1 mL medium, DNA: Trans IT X2® were mixed in the ratio of 1 : 2 (3 μg/6 μL) in antibiotic free medium and added to the cells within 20 min.

Publication protocol

TF‐1 cells (CRL‐2003; ATCC; Manassas, VA; RRID:CVCL_0559), an erythroleukemic cell line from human blood, were maintained at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mM sodium pyruvate, 4.5 g/L glucose and 2 ng/mL GM‐CSF (PHC2015). Prior to induction of differentiation, cells were kept in medium without GM‐CSF overnight, followed by the addition of EPO (1 U/mL; PHC2054) on the next day. The TF‐1 cell line is not listed in the database of commonly misidentified cell lines (International Cell Line Authentication Committee), and not authenticated by the authors. All culture reagents were from Life Technologies (Life Technologies, Carlsbad, CA, USA). The renewal of medium and EPO (1 U/mL) were done on alternative days, and the cell density was maintained at 4 × 105 cells/mL. Cultured TF‐1 cells were transfected with cDNA constructs with Trans IT X2® Dynamic Delivery System (Mirus BIO, Madison, WI, USA) for AChE shRNA and (Santa Cruz, Dallas, TX, USA). Transfection was performed 1 day after cells were plated. For every 5 × 106 cells in 1 mL medium, DNA: Trans IT X2® were mixed in the ratio of 1 : 2 (3 μg/6 μL) in antibiotic free medium and added to the cells within 20 min.

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Manufacturer protocol

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