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.
4. Trypsinise for 3-5 minutes with 2 ml trypsin.
5. Stop trypsinisation using 6 ml DMEM.
6. Triturate 30 times.
7. Centrifuge on a table-top centrifuge at 700 rpm for 5 min at room temperature.
8. Throw away supernatant (this step gets rid of the trypsin)
9. Resuspend in 10 ml DMEM and triturate 30 times.
10. Transfer 5 ml in a new tube and add 45 ml DMEM.
11. 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