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

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.