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

Transfection Lipofectamine 3000

• Seed cells to be 70–90% confluent at transfection
• Dilute Lipofectamine Reagent in Opti-MEM Medium and mix well
• prepare mastermix of DNA (0,5 - 5 μg/μl) by diluting DNA in Opti-MEM Medium, then add P3000 (2 μl/μg DNA) Reagent and Mix wel
• Add diluted DNA to each tube of diluted Lipofectamine Reagent (1:1 ratio)
• Incubate for 5 minutes at room temperature
• Add DNA-lipid complex to cells
• Visualize/analyze transfected cells. Incubate cells for 2–4 days at 37°C. Then, analyze transfected cells.


• for more information: see thermofisher.com

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

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.

PCR

Reaction mixture

Product	Final concentration	Volume for 50µL reaction
5x Phusion buffer HF	1x	10µL
dNTPs (10mM)	200µM each	1µL
Forward primer (25µM)	0.5µM	1µL
Reverse primer (25µM)	0.5µM	1µL
Template DNA		1µL
Phusion DNA polymerase	0.02U/µL	0.5µL
Milli-Q Water		35.5µL

Protocol

Step	Time	Temperature	Number of cycles
Initial denaturation	30’’	98ºC	1
Denaturation	10’’	98ºC	30
Annealing	30’’	*ºC (*: 2 degrees below Tm)
Extension	3’30’’	72ºC
Final extension	7’	72ºC	1
End	-	4ºC

Patch Clamp

Methods

• We used whole-cell patch clamp to measure the membrane voltage and ion currents across a HEK cell membrane. To measure whole-cell, we pushed a glass pipette against the membrane of a cell, to seal the membrane completely against the membrane. Afterwards, we caused a sudden negative pressure in the glass pipette, which destroys the membrane in the opening of the glass pipette. The solution within the pipette will subsequentially mix with the intracellular compartment of the cell, which allows us to measure with a pre-defined intracellular buffer.

Voltage clamp

• To study the currents through different ion channels with whole-cell patch clamp, we used a specific technique called "Voltage clamp". Here, we maintain a pre-defined membrane potential to study the activation of α1G, HCN2 and hERG. Each of these ion channels has a specific way of activation, which can be quantified using voltage clamp. When clamping the voltage at a certain value, a cell will adapt to the voltage by increasing or decreasing ion currents. These currents will be measured using this technique

Current clamp

• When trying to measure an oscillation in the cell membrane with patch-clamp, you cannot use voltage-clamp, since this technique doesn't allow the cell to change its membrane potential. We used current-clamp instead. Here, you can inject a pre-defined current into the cell which allows you to see how the membrane potential of the cell responds to the current. When a cell oscillates by itself, the membrane potential will oscillate while injecting 0 current. However, we often needed to inject a small negative current (-300pA) into a cell to elicit an oscillation in a cell with an extracellular Krebs solution.

Calcium imaging

• Calcium imaging is a scientific technique which can be used to measure and follow the calcium ions inside and/or outside the cell. We used a specific dye (Fura2-AM) to follow the calcium ions by the rate of fluorescence. For our project, we only used the calcium imaging for testing whether there were oscillations or not and what potassium concentrations (in a krebs buffer) were needed. We did not mathematically analyse the data we received. We only used it as a criteria for the patch clamp.

Preparation of 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

Intra- and extracellular buffers

Buffers for stable HEK-HCN cells

External Solution	MW (g/mol)	mM	g/l
NaCl	58,44	150	8,766
KCl	74,56	10	0,7456
CaCl2 x 2H2O	147,02	3	0,44106
MgCl2 x 6H2O	203,3	1	0,2033
Hepes	238,31	10	2,3831
pH 7.4 NaOH

Pipetsolution	MW (g/mol)	mM	g/100 ml
KCl	74,56	150	1,1184
MgCl2 x 6H2O	203,3	0,5	0,010165
Hepes	238,31	10	0,23831
EGTA	380,35	1	0,038035
pH 7.4 KOH

Activator HCN: 100 µM cAMP MM 329,21 g/mol	Stock 30 mM: 0,01g/ml
Blokker HCN: 10 mM CsCl2 MM 168,37 g/mol	Stock 1 M: 0,168 g/ml

Buffers for transfected HEK-HCN and HEK-hERG cells

Krebs	MW (g/mol)	mM	g/l
NaCl	58,44	150	8,766
KCl	74,56	5	0,3728
CaCl2 x 2H2O	147,02	2	0,29404
MgCl2 x 6H2O	203,3	1,5	0,30495
Hepes	238,31	10	2,3831
Glucose monohydrate	198,17	10	1,9817
pH 7.4 NaOH

K-Sol	MW (g/mol)	mM	g/l
NaCl	58,44	0	0
KCl	74,56	155	11,5568
CaCl2 x 2H2O	147,02	2	0,29404
MgCl2 x 6H2O	203,3	1,5	0,30495
Hepes	238,31	10	2,3831
Glucose monohydrate	198,17	10	1,9817
pH 7.4 NaOH

• The Na⁺ and K⁺ concentration is always 155 mM and when we made another buffer with another K ⁺-concentration, the Na ⁺ concentration changes with it. The other components remained the same. (The most useful K ⁺ concentration is between 1 and 5 mM.)

Pipetsolution	MW (g/mol)	mM	g/100 ml
KCl	74,56	150	1,1184
Hepes	238,31	10	0,23831
MgCl2 x 6H2O	203,3	0,5	0,010165
EGTA	380,35	1	0,038035
pH 7.4 with KOH

Buffers for TRPM3 cells

external solution	MW (g/mol)	mM	g/100 ml
NaCl	58,44	150	8,77
MgCl2 x 6H2O	203,3	1	0,2
Hepes	238,31	10	2,38
pH 7.4 with NaOH

Pipet solution	MW (g/mol)	mM	g/100 ml
dl-Asp	133,1	100	1,331
CsCl	168,36	45	0,757
Hepes	238,31	10	0,238
MgCl2 x 6H2O	203,3	1	0,0203
EGTA	380,35	10	0,38
pH 7.4 with CsOH

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