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:
    1. Pipet 298 ul OptiMEM in a 1.5 ml eppendorf tube labelled “P” (“P” stands for “plasmid”)
    2. Pipet 288 ul OptiMEM in a 1.5 ml eppendorf tube labelled “L” (“L” stands for “Lipofectamine-2000”)
    3. Pipet 2 ul pDNA in the “P” tube.
    4. 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.

  1. Warm up medium, PBS and trypsin in the water bath.
  2. Wash cells with 10 ml PBS.
  3. Trypsinise for 3-5 minutes with 2 ml trypsin.
  4. Stop trypsinisation using 6 ml DMEM.
  5. Triturate 30 times.
  6. Centrifuge on a table-top centrifuge at 700 rpm for 5 min at room temperature.
  7. Throw away supernatant (this step gets rid of the trypsin)
  8. Resuspend in 10 ml DMEM and triturate 30 times.
  9. Transfer 5 ml in a new tube and add 45 ml DMEM.
  10. Pipet 2 ml in 3.5 cm diameter dishes for patch-clamp experiments the following day.
  11. 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

Intracellular (pipette) solution for whole-cell 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.


Chimerel et al., Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells, Cell Rep., 2014
Link to the article in PubMed

Extracellular (bath) solution for whole-cell patch-clamp in GLUTag cells

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.


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