Medium of HEK 293 cells
- DMEM (Gibco N° 41965-039) 500 ml
-
- 50 ml (10 %) FCS = (Hyclone)
- 10 ml Penicillin/Streptomycin (Gibco N° 15070-071) = 100-100U/ml
- 10 ml Glutamax (Gibco N° 35050-038) = 10 mM
- 5 ml Non-Essential-Amino-Acids (Gibco N° 1140-035) = 10x
Growing Conditions
- Incubate at 37°C and 10% CO2
- Split cells around 70-90% confluency
- Change medium twice a week
Standard procedure for Trypsinization
- wash the cells with Versene (Gibco N° 15040-033, ± 2 min)
- Remove versene and add Trypsin (Gibco N° 25300-096)
- Incubate at 37°C (around 3-5 min)
- Stop the trypsinization by adding 5-10 times volume of medium
- Count cells
- Centrifuge for 5 minutes at 1200 rpm
- Aspirate supernatant and resuspend the cells in medium
- Plate cells for experiments or culture
Coating of Glass coverslips (for patch-clamp or Ca2+ imaging)
Poly-l-lysine (PLL, sigma P2636)
Dissolved at 0.1 mg/ml in Milli-Q H2O
Stored at –20°C
Once opened stored at RT
Coverslips
18mm
- Prepare 12 well plates with a coverslip in each well
- Transfer 1 sterilized coverslip to each well
- Add 350 ml of PLL to each coverslip
- Incubate for 20 minutes at RT
- Aspirate and add Milli-Q H2O to each coverslip
- Incubate for 20 minutes at RT
- Aspirate H2O and store for experiments
You can keep the plates for several days at 4°C
Seeding Cells
Seeding cells for single-cell experiments
- Spray your hands with ethanol, take the 6 well plate with cells out of the incubator and check the density of the cells
- Place the cells under the flow
- Remove the medium
- Wash the cells with 1 ml of versene for 2 minutes
- Remove versene and add 500μl of trypsin
- Incubate at 37°C, 10% CO2 for about 5-10 minutes
- Stop the trypsinization by adding 5 ml of medium.
- Transfer cell suspension to a 50 ml Falcon tube
- Separate the cells by pulling them with a 5 ml syringe through a black needle of 22G (5-10 times)
1.1 Ca2+ imaging
Cells are typically seeded in a 12-well plate, on PLL coated round coverslips of 18 mm
Add 1 ml of medium to each 12-well of your plate
If the cells in the 6-well had a density of 95-100%, typically seed 400-500 microliters of cell suspension per 12-well (density depending on experimental needs)
Incubate at least 2 hours for cell attachment to the coverslip
1.2 Patch clamp
Cells are typically seeded in a 12-well plate, on PLL coated round coverslips of 18 mm
Add 1 ml of medium to each 12-well of your plate
If the cells in the 6-well had a density of 95-100%, typically seed 100-300 microliters of cells per 12-well (density depending on experimental needs)
Incubate at least 2 hours for cell attachment to the coverslip
Ca2+ imaging
Fura-2, AM (MW: 1001,9)
Dilute 50 μg of Fura-2 in 50 μl DMSO (1 mM stock solution)
Make aliquots of 2 μl in brown eppies and store at -20°C
To load cells (1 well): add 2 μl of Fura-2 to 1 ml of medium (2μM work solution)
Incubate cells for 30 minutes before measurement
