Ingredients
- agarose
- potassium chloride (KCl; MW = 74.54 g/mol)
- glass capillaries
- ethanol burner
- syringe
Procedure
- Prepare bent capillary tubes. Alight an ethanol burner by dipping the wick in EtOH, e.g. filling a 1.5 ml tube (eppendorf) with ethanol and dipping the wick in it. Hover the capillary (at 1/3 of its length) over the fire and keep pushing on the short end with a pen until the capillary is bent at a right angle.
- Prepare a 20 ml solution at 1% agarose. Weight 0.2 g agarose and dissolve it in 20 ml ddH2O in a 50 ml Falcon tube.
- Weight the appropriate amount of KCl in order to achieve a final concentration of 3 M. I need:
0.020 l x 3 mol/l = 0.06 mol KCl
0.06 mol x 74.54 g/mol = 4.4724 = 4.47 g KCl
DO NOT ADD THE KCl TO THE AGAROSE SOLUTION BEFORE MICROWAVING THE AGAROSE!!! (COULD CAUSE SPARKS IF KCl IS MICROWAVED)
- Microwave the agarose solution.
- Add the KCl and dissolve it.
- Aspire the solution with the syringe.
- Fill bent capillary tubes with the solution. Tip: hold the connection between capillary and syringe in order to prevent the mixture from spilling around the capillary. Quickly dry capillaries. Trim the ends of capillaries with a diamond cutter.
Capillaries should not contain any bubbles.
Storage
Store the capillaries, i.e. agar bridges, at room temperature in a 3 M KCl solution.
Day 1: split cells
Matrigel 6 cm diameter Petri dishes:
- Dilute matrigel 100-fold with DMEM without any additives
- Pipet 3 ml of diluted matrigel on a 6 cm diameter Petri dish
- Incubate for 30 min at 37°C
During the incubation, trypsinise a T75 flask of GLUTag cells and resuspend them in 10 ml medium (50/50 fresh/conditioned media).
Remove the matrigel from the dish and add 4 ml full DMEM medium.
Add 2.5 ml of the resuspension in the dish and incubate overnight at 37°C/5% CO2.
Day 2: transfect cells using Lipofectamine-2000
- 1 hour before transfection, replace the full DMEM medium. Don’t forget to warm the medium in the 37°C water bath.
- Prepare the plasmid and Lipofectamine-2000 seperately in OptiMEM. Use 3 ug of plasmid and 12 ul of Lipofectamine-2000. Eg. if the plasmid concentration is 1.5 ug/ul:
- Pipet 298 ul OptiMEM in a 1.5 ml eppendorf tube labelled “P” (“P” stands for “plasmid”)
- Pipet 288 ul OptiMEM in a 1.5 ml eppendorf tube labelled “L” (“L” stands for “Lipofectamine-2000”)
- Pipet 2 ul pDNA in the “P” tube.
- Pipet 12 ul Lipofectamine-2000 (straight from the fridge) in the “L” tube. Do not mix the lipofectamine-2000 by pipetting up and down. Lipofectamine is sticky and will stick to the pipette if this is done!
- Let the two tubes sit for 10 min in the hood.
- Add the tube “P” content to the tube “L” (never do the reverse, the less you pipette Lipofectamine-2000, the better)
- Incubate for 20 min in the hood
- Add the 600 ul mixture to the 6 cm diameter dish dropwise.
- Incubate overnight at 37°C/5% CO2.
Transfer transfected cells into smaller recording dishes
This step is necessary in order to go from a confluent layer of cells to single cells scattered on a dish that are amenable to being patch-clamped.
- Warm up medium, PBS and trypsin in the water bath.
- Wash cells with 10 ml PBS.
-
- Trypsinise for 3-5 minutes with 2 ml trypsin.
- Stop trypsinisation using 6 ml DMEM.
- Triturate 30 times.
- Centrifuge on a table-top centrifuge at 700 rpm for 5 min at room temperature.
- Throw away supernatant (this step gets rid of the trypsin)
- Resuspend in 10 ml DMEM and triturate 30 times.
- Transfer 5 ml in a new tube and add 45 ml DMEM.
- Pipet 2 ml in 3.5 cm diameter dishes for patch-clamp experiments the following day.
Each dish contains 0.5 x 2/50 = 0.5 x 1/25 = 0.5 x 0.04 = 0.02 = 2 % of the initial cells. It is advisable to make another two-fold dilution to get 1% of the initial cells on a few dishes in case the former dilution is not strong enough.
Day 4: patch-clamp
This solution will be in dialysis with the cytosol and hence is similar to it in composition (high potassium and low sodium).
| Chemical |
mM |
MW/concentration |
for 100 ml |
| KCl |
107 |
74.55 |
0.7976 g |
| CaCl2 |
1 |
1 M |
100 ul |
| MgCl2 |
7 |
1 M |
700 ul |
| EGTA |
11 |
380.35 |
0.4183 |
| HEPES |
10 |
238.3 |
0.2383 g |
| Na2ATP |
5 |
569.16 |
0.2846 |
Adjust to pH to 7.2 using potassium hydroxide (KOH).
Always check the molecular weight on the bottles of the chemicals. It may vary, due to different water contents for example.
Reference
Chimerel et al., Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells, Cell Rep., 2014
Link to the article in PubMed
This solution is needed to keep cells in a physiological environment during patch-clamp experiments. It also contains glucose as a nutrient.
| Chemical |
mM |
MW/concentration |
for 1 L |
for 500 ml |
for 500 ml 10X |
| NaCl |
138 |
58.44 |
8.065 g |
4.0325 g |
40.325 g |
| KCl |
4.5 |
74.55 |
0.335 g |
0.1675 g |
1.675 g |
| NaHCO3 |
4.2 |
84.01 |
0.3528 g |
0.1764 g |
1.764 g |
| NaH2PO4 |
1.2 |
120 |
0.144 g |
0.072 g |
0.72 g |
| CaCl2 |
2.6 |
1 M |
2.6 ml |
1.3 ml |
– |
| MgCl2 |
2.6 |
1 M |
1.2 ml |
0.6 ml |
6 ml |
| HEPES |
10 |
238.3 |
2.382 g |
1.1915 g |
11.915 g |
Glucose: 1 mM (18 MG/100 ml)
pH 7.4 using NaOH
For a 500 ml 1X solution, I added 2.4 ml of NaOH at 1 M concentration.
Add CaCl2 and adjust the pH when the solution concentration is 1X.
Add glucose to the solution needed on the day, usually 50 ml.
Always check the molecular weight on the bottles of the chemicals. It may vary, due to different water contents for example.
Reference
Rogers et al., Electrical activity-triggered glucagon-like peptide-1
secretion from primary murine L-cells, J. Physiol., 2011
Link to the article in PubMed
Activités scientifiques de Cristian Riccio