Team:KU Leuven/Protocols

Protocols

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.

Cell Culture

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

Wet Lab

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.

Electrophysiology and calcium imaging

Patch clamp

Extracellular buffers

• 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.

Calcium imaging

Extracellular buffers

• For both patch-clamp and calcium imaging, we used different extracellular potassium concentrations, since the resting membrane potential of cells mostly depend on the extracellular potassium concentration.

Buffers

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