Experiments were performed using HEK293T cells (ATCC, Manassas, VA, USA), which were grown in Dulbecco’s modified Eagle’s medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal calf serum (ATCC, Manassas, VA, USA), 50 U/mL penicillin, and 50 μg/mL streptomycin (Sigma, St Louis, MO, USA) in a humidified 5% CO2 atmosphere at 37 °C. The cells were cultured in 75 cm2 plastic culture flasks (NUNC, Rochester, NY, USA) for 36–72 h until they reached 80–95% confluence. On the day before transfection, ~150,000 cells were seeded on 35 mm culture dishes (Sarstedt, Newton, NC, USA) and incubated at 37 °C for at least 24 h. Transfection was performed using 2 μg of DNA and 2 μL of jetPRIME reagent in 2 mL of Dulbecco’s modified Eagle’s medium according to the manufacturer’s instructions (PolyPlus-transfection, Illkirch, France). After 24 h of incubation, the transfected cells were mechanically dispersed and reseeded on 35 mm Corning 3294 CellBIND Surface cell culture dishes (Corning, City of Corning, NY, USA) for 2–8 h prior to recording. For the dye uptake measurements, the transfected cells were plated on 12 mm poly-L-lysine-coated coverslips (Glaswarenfabrik Karl Hecht KG, Sandheim, Germany).
75 cm2 plastic culture flasks
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 75 cm2 plastic culture flasks are a standard laboratory equipment used for culturing cells in vitro. These flasks provide a consistent and controlled environment for the growth and maintenance of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
35 mm culture dishes, by Sarstedt (4 mentions)
Jetprime reagent, by Polyplus Transfection (3 mentions)
Model ix71, by Olympus (3 mentions)
Hek293t cell, by American Type Culture Collection (3 mentions)
3294 cellbind surface, by Corning (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
4 protocols using 75 cm2 plastic culture flasks
1
HEK293T Cell Transfection Protocol
2
Transfection of HEK-293T cells for P2Y receptor studies
3
Recombinant Channel Expression in HEK 293T Cells
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To express the recombinant channels, we used human embryonic kidney (HEK) 293T cells (American Type Culture Collection, Rockville, MD, USA) grown in Dulbecco modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO), 50 U/ml penicillin and 50 µg/ml streptomycin (both Thermo Fisher Scientific, Waltham, MA) in a humidified 5% CO2 and 95% air at 37°C. Cells were cultured in 75 cm2 plastic culture flasks (NUNC, Rochester, NY) for 36–72 hours until reaching 80–95% confluence. Before the day of transfection, the cells were plated on 35 mm culture dishes (Sarstedt, Newton, NC) and incubated at 37°C for at least 24 h. Transfection was done using 2 µg of either WT or mutant receptor DNA with 2 µl of JetPrime reagent in 2 ml of Dulbecco modified Eagle’s medium, according to manufacturer’s instructions (PolyPlus-transfection, Illkirch, France). Transfected cells were identified by the fluorescence signal of EGFP using the Olympus IX71 inverted research microscope with fluorescence illuminators (Model IX71; Olympus, Melville, NY).
4
Transient Transfection of HEK293 Cells
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Experiments were performed on human embryonic kidney 293 cells (HEK293; American Type Culture Collection), which were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin in a humidified 5% CO2 atmosphere at 37°C. Cells were cultured in 75-cm2 plastic culture flasks (NUNC, Rochester, NY, USA) for 36–72 h until they reached 80–95% confluence. Before the day of transfection, ~150 000 cells were plated on 35 mm culture dishes (Sarstedt, Newton, NC, USA) and incubated at 37°C for at least 24 h. For each culture dish of HEK293 cells, transfection of wild-type P2X4R was conducted using 2 μg of DNA with 2 μl of jetPRIME reagent in 2 ml of Dulbecco modified Eagle’s medium, according to the manufacturer’s instructions (PolyPlus-transfection, Illkirch, France). After 24–48 h of incubation, the transfected cells were mechanically dispersed and re-cultured on 35 mm dishes of Corning 3294 CellBIND Surface for 1–4 hours before recording. Transfected cells were identified by the fluorescence signal of enhanced green fluorescent protein using an inverted research microscope with fluorescence illuminators (Model IX71; Olympus, Melville, NY).
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