Experiments

In the lab, we used different experimental procedures. There are protocols for the wet and bacterial lab, for the cell culture lab and for the electrophysiology lab.

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

Team:KU Leuven/Protocols

Experiments

Cell Culture

Cell Culture

Medium of HEK 293 cells

Standard procedure for Trypsinization

Coating of Glass coverslips (for patch-clamp or Ca2+ imaging)

Seeding Cells

Seeding cells for single-cell experiments

1.1 Ca²⁺ imaging

1.2 Patch clamp

Ca²⁺ imaging

Fura-2, AM (MW: 1001,9)

Experiment 2: Andere korte beschrijving

Team:KU Leuven/Protocols

Experiments

Cell Culture

Cell Culture

Medium of HEK 293 cells

Standard procedure for Trypsinization

Coating of Glass coverslips (for patch-clamp or Ca2+ imaging)

Seeding Cells

Seeding cells for single-cell experiments

1.1 Ca2+ imaging

1.2 Patch clamp

Ca2+ imaging

Fura-2, AM (MW: 1001,9)

Experiment 2: Andere korte beschrijving

1.1 Ca²⁺ imaging

Ca²⁺ imaging