Plasmids were transfected into 293T HEK cells (ATCC CRL-3216) at roughly 80% confluency in 10 cm tissue culture plates coated with poly-d-lysine using Lipofectamine 3000. 293T HEK cells were not further authenticated or tested for mycoplasma. The lipofectamine media was exchanged 16 hours later, and the viral supernatant was collected at 72h post-transfection. The collected viral supernatant was filtered via a 0.45 μm filtration unit, and concentrated using the LentiX concentrator (Takara) for 2 hours at 4 C and then spun down at 1500 x g for 45 minutes at 4 C. The concentrated supernatant was subsequently aliquoted, flash frozen, and stored in −80°C until use.
293t hek cells
Manufactured by American Type Culture Collection
Sourced in United States
293T HEK cells are a commonly used human embryonic kidney cell line derived from human embryonic kidney cells. They are known for their high transfection efficiency and are widely used in biological research and biotechnology applications.
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5 protocols using 293t hek cells
1
Lentiviral Vector Production in 293T Cells
2
Cell Lines for Protein and Virus Studies
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HEK293T cells used for protein purification, virus packaging and stable cell line generation, and AMPKα1/2 double knockout (DKO) MEF cells used for stable cell line generation were cultured with DMEM high glucose medium (HyClone, SH30243.01), supplemented with 10% FBS (Gibico, 10099–141) and 1% Penicillin/Streptomycin (HyClone SV30010). 293T HEK cells were obtained from ATCC, not authenticated, and mycoplasma negative. AMPKα1/2 double knockout (DKO) MEF cells were provided by Dr. Benoit Viollet, validated by immunoblotting and determined as mycoplasma negative.
3
Scalable Production of rAAV Vectors
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293T HEK cells (ATCC CRL 3216, American Type Culture Collection, Manassas, VA, USA) cultured in 4 10-floor cell factories until ~85% confluent. Cells were triple transfected with Rep2Cap9 (Serotype 2 Rep proteins, Serotype 9 capsid proteins), pHelper (Adenovirus helper constructs), and scAAV-CBA-SNCA using 25 kDa Polyethyleneimine (PEI) at a molar ratio of 1:1:1. Media was changed 24 hours after transfection, and cells were harvested at 48 hours after transfection. Cells were suspended in 10 mmol Tris, pH = 8.0, lysed by 5 freeze-thaw-cycles in liquid nitrogen, DNAse treated, and protease treated. CsCl crystals were added to the lysate (0.631 g of CsCl per ml of the lysate) to generate a solution with a density of ∼1.4 mg/ml. After incubation at 37°C for 45 min, the solution was centrifuged at 4000 rpm in an Eppendorf 5810 R at 4°C. Virus was purified from lysate by 3 rounds of density gradient centrifugation at an average RCF of 158,000. High titer fractions were detected after each round of centrifugation using quantitative real-time PCR. The final fractions were dialyzed exhaustively against phosphate buffered saline and stored at 4°C until use.
4
EMSA Analysis of Transcription Factor Binding
5
Production and Purification of AAV Vectors
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Viral production was performed as previously described.29 Briefly, 293 T HEK cells (ATCC CRL 3216) were cultured in four 10‐floor cell factories until approximately 85% confluent. Cells were triple transfected with Rep2Cap9 (serotype 2 Rep proteins, serotype 9 capsid proteins), pHelper (adenovirus helper constructs; Stratagene), and scAAV‐CBA‐myomixer using 25 kDa polyethyleneimine at a molar ratio of 1:1:1. Cells were harvested at 48 h after transfection and suspended in 10 mmol Tris, pH = 8.0, lysed by five freeze–thaw cycles in liquid nitrogen, DNAse treated, and protease treated. CsCl crystals were added to the lysate (0.631 g of CsCl per mL of the lysate) to generate a solution with a density of approximately 1.4 mg/mL. After incubation at 37°C for 45 min, the solution was centrifuged at 3184 g in an Eppendorf 5810 R at 4°C. Virus was purified from lysate by three rounds of density gradient centrifugation at an average RCF of 158 000. Vector titres were determined by quantitative real‐time PCR. The final fractions were dialyzed against PBS and stored at 4°C until use.
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