Tag Archives: ephys

Making agar bridges for electrophysiology


  • agarose
  • potassium chloride (KCl; MW = 74.54 g/mol)
  • glass capillaries
  • ethanol burner
  • syringe


  1. 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.
  2. 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.
  3. 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

  5. Microwave the agarose solution.
  6. Add the KCl and dissolve it.
  7. Aspire the solution with the syringe.
  8. 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.
  9. Capillaries should not contain any bubbles.


    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:
    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