CRL-3479 ™
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
Description: The tet-on spiking HEK cells constitutively express a voltage-gated sodium channel NaV1.5, and inducibly (tet-on) express Kir2.1. When grown in a confluent monolayer these cells can support propagation of action potentials through the culture. Action potentials can also be triggered by electrical stimulation.
Cell line creation
First, NaV1.5-Puro+ HEK293T cells were generated as previously described1. The tet-inducible expression system was designed by the Eric Campeau lab and obtained through Addgene. Kir2.1_CFP was cloned into the open reading frame of pLenti-CMVtight-eGFP-Neo vector (Addgene Plasmid #26586). pLenti-CMV-rtTA3-Blast (Addgene Plasmid #26431) was used directly to package lentivirus. NaV1.5-Puro+ HEK293T cells were simultaneously infected with pLenti-CMV-rtTA3-Blast and pLenti-CMVtight-Kir2.1_CFP-Neo. The cells were first selected with three antibiotics (2 μg/mL puromycin, 5 μg/mL blasticidin, 200 μg/mL Geneticin/G418), then induced with doxycycline (2 μg/mL) for ~30 hours before FACS purification based on the CFP marker. The CFP+ cells were seeded into 96-well plates (1 cell per well) and cultured 3~4 weeks under standard conditions for HEK cell culture. The expanded monoclonal cells were screened with current clamp.
Expanding and culturing
Upon receiving the cells, store them in liquid nitrogen or directly revive and grow the cells in DMEM10 (1L DMEM, 100 mL FBS, 10 mL GlutaMax-I (100x), and 10 mL pen/strep (100x). When growing up these cells for the first time, freeze down multiple vials and store them in liquid nitrogen. Start a new vial after approximately 10 passages. Older cells may give less consistent results. When thawing a new vial, do not include antibiotics from the very beginning. After the first or second passage, when the cells have regained a healthy HEK cell morphology, it is recommended to include antibiotics in the growth medium to maintain genetic stability (2 μg/ml puromycin, 5 μg/ml blasticidin, 200 μg/ml Geneticin/G418). That the cell line is reasonably stable without these antibiotics.
Culturing spiking HEK cells for spontaneous or stimulated spikes
HEK cells do not tolerate sustained Kir2.1 expression, so maintain the cells on a master plate without doxycycline addition. The cells grow well under culture conditions suitable for wild-type HEK293T cells, but at a lower growth rate. Grow the cells on tissue culture-treated plates.
For imaging applications that require glass-bottomed dishes, coat the dish with poly-D-lysine (PDL) before use. Typically, we seed 400-500k tet-on spiking cells in the well of a 14-mm glass-bottomed dish. The number should be adjusted based on the surface area of the glass. Seed the cells evenly and avoid introducing air bubbles to the bottom of the dish. Be sure to supply doxycycline+ (1~2 μg/mL) medium to induce Kir2.1 expression on Day 0. The cells need to form a monolayer to reliably spike. Typically use the cells for imaging 36 hours to 72 hours after seeding. However, if the monolayer is overgrown and overcrowded the cells may become "silent". Large clusters of spiking HEK cells may spike in response to stimulation, but monolayer cultures give the most consistent results.
The complete medium for this cell line is Dulbecco's Minimal Essential Medium (DMEM; ATCC 20-2002). To make the complete medium add the following components:
To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.
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