Medium of HEK 293 cells

Medium contents

• 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. The plates can be kept 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)

I. Ca²⁺ imaging

• Cells are typically seeded in a 12-well plate with 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

II. 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

Transformation of MAX Efficiency™ DH5α™ Competent Cells

Day 1

• Add 25µL of bacteria to the bottom of a polypropylene 14mL Falcon tube (352059). Place on ice.
• Add either 0.5µL of 100ng/µL plasmid or 1 to 5µL of a ligation mix to the bacteria. Mix gently by shaking. Leave on ice for 30 minutes
• Preheat the SOC medium and waterbath to 42ºC.
• Perform a heat shock. Place cells at 42ºC for 30 to 45 seconds. Return to ice for 2 minutes.
• Add 0.5mL SOC medium.
• Place in shaking incubator at 37ºC for 1 hour.
• Plate and spread on plates containing 50µg/mL of a specific antibiotic resistance markers. Incubate overnight at 37ºC for maximum 16 hours.

Day 2

• Add 3mL of LB medium containing 50µg/mL of a specific antibiotic resistance marker to a 14mL polystyrene Falcon tube (352057).
• Pick a single colony using a Inoculation loop. Transfer colony into medium and shake. In order to perform a miniprep, grow at 37ºC overnight and skip to 'Day 3'. Otherwise, grow for 3 to 4 hours and continue.
• Add 70mL LB medium containing 50µg/mL of a specific antibiotic resistance marker to a sterile erlenmeyer.
• Add the bacteria to the new medium: ½ of the medium in the Falcon if the growth is visible, all of the medium if the solution is still clear.
• Place erlenmeyers in shaking incubator at 37ºC overnight or for maximum 16 hours.

Day 3

• Harvest plasmids using mini-, midi- or maxiprep kit.