HeLa cells (derived from Cell Bank of Chinese Academy of Science) and HEK293T cells (derived from Invitrogen) were grown in high glucose DMEM (Hyclone) with 10% FBS (Gibco). All cell lines were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2. All cell lines were tested for mycoplasma contamination and experiments were conducted under mycoplasma- negative conditions.
For transient transfection, approximately 60,000 or 20,000 HeLa cells were plated on a 4-well glass bottomed dish (35 mm2) or 96-well plate per well (WHB), respectively. The plasmids were transfected with lipofectamine 2000 (Invitrogen) or Hieff Trans (YEASEN, China) according to the manufacturer’s protocol.
The pLVX lentiviral plasmids encoding sensors (iNap, roGFP1, FLII12 (link)Pglu700µ), NADK were constructed. Lentivirus was produced by co-transfecting two lentiviral packaging vectors (pMD2G and psPAX2) in HEK293T cells. Lentiviral supernatants were collected 48 and 72 hr after transfection. HeLa cells in 6-well tissue culture plates were infected in media containing 4 µg/ml polybrene and centrifuged at 1,800 rpm for 1 hr. Post-infection, virus was removed and cells were selected with 0.2–1 µg/ml puromycin for 1 week. After 1 week, the stable cells were selected by FACS Aria I (BD).
For transient transfection, approximately 60,000 or 20,000 HeLa cells were plated on a 4-well glass bottomed dish (35 mm2) or 96-well plate per well (WHB), respectively. The plasmids were transfected with lipofectamine 2000 (Invitrogen) or Hieff Trans (YEASEN, China) according to the manufacturer’s protocol.
The pLVX lentiviral plasmids encoding sensors (iNap, roGFP1, FLII12 (link)Pglu700µ), NADK were constructed. Lentivirus was produced by co-transfecting two lentiviral packaging vectors (pMD2G and psPAX2) in HEK293T cells. Lentiviral supernatants were collected 48 and 72 hr after transfection. HeLa cells in 6-well tissue culture plates were infected in media containing 4 µg/ml polybrene and centrifuged at 1,800 rpm for 1 hr. Post-infection, virus was removed and cells were selected with 0.2–1 µg/ml puromycin for 1 week. After 1 week, the stable cells were selected by FACS Aria I (BD).
