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
Starting point: confluent T75 flask containing stuck GLUTag cells and 20 ml DMEM medium
All steps are undertaken in a hood!
Day 1: Splitting a confluent flask
- Label one 50 ml falcon tube with “condition” and one with “rubbish”
- Transfer the DMEM from the flask into the falcon labelled “condition”
- Wash cells with 10 ml PBS and transfer the dirty PBS into the “rubbish” tube
- Add 3 ml trypsin to the flask and leave it in the incubator for 3-5 min
- Check under the microscope that the cells have detached
- Add 5 ml fresh medium to stop the trypsinisation reaction
- Spin the cells down at 600 rpm for 5 min
- Chuck the supernatant
- Resuspend the cells in 5 ml fresh medium and triturate them 20 times with a 5 ml stripette (Note: using a 5 ml stripette rather than a bigger-volume one will help separate the cells due to the narrower aperture of the pipette)
- Add 5 ml condition medium from the “condition” tube to the resupsended cells
- Add 3 ml of the resuspension to a new T75 flask containing 17 ml fresh DMEM medium
- The other 7 ml can be used to seed Petri dishes for use in experiments, e.g.: calcium imaging, electrophysiology, secretion experiments, transfection, etc.
Day 3: Changing medium
Exchange 10 ml supernatant for 10 ml fresh DMEM medium
Day 5: Splitting a confluent flask
Refer to Day 1.
Cristian Riccio, Institute of Metabolic Science, University of Cambridge
Acknowledgments
Thanks to Dr. Edward Emery and Dr. Frank Reimann from the Institute of Metabolic Science for their contribution to the establishment of this protocol.
Day 1 : split cells
Starting point: T75 flask with GLUTag cells at a confluence > 80%
End point: 10 cm diameter Petri dish with GLUTag cells
- Transfer 5 ml of condition medium into a falcon tube.
- Throw away the rest of the condition medium (should be 15 ml)
- Wash cells with 10 ml PBS
- Add 3 ml trypsin and incubate for 5 min in the 37°C/5% CO2 incubator (check that cells are detached at the end of this short incubation and, if not, tap on the side of the flask to detach cells)
- During the trypsinisation, add 2 ml complete DMEM to the 5 ml condition medium
- At the end of the trypsinisation, add the 7 ml medium and triturate the cells. The total volume of cells is now 10 ml
- Add 6 ml complete DMEM to each of two 10 cm diameter Petri dishes
- Add 4 ml of resupspended cells to each of the Petri dishes
- Add the remaining 2 ml of resuspended cells to a new T75 flask containing 18 ml complete DMEM
- Incubate overnight in the 37°C incubator containing 5% CO2
Day 2 : Transfection day
Starting point:
- 10 cm diameter Petri dish with attached GLUTag cells x 2
End point:
- 10 cm diameter Petri dish with transfected GLUTag cells x 1
- 10 cm diameter Petri dish with non-transfected GLUTag cells x 1
Reagents
- Transfection agent: Lipofectamine-2000
- plasmidic DNA
- Optimem
Initial and final conditions
- Initial medium volume: 10 ml complete DMEM
- Final medium volume: 6 ml complete DMEM + 1.5 ml Optimem
- Initial pDNA concentration: 0
- Final pDNA concentration: 0.04 ug/cm^2, i.e. 3 ug/75 cm^2
- Initial Lipofectamine-2000 concentration: 0
- Final Lipofectamine-2000 concentration: 12 ul/ug pDNA, i.e. 36 ul/75 cm^2
Parameters
pDNA stock concentration: 2 ug/ul
Procedure (in hood)
- Pipet 750 ul of Optimem in each of two eppendorf tubes
- Pipet 3 ug/(2 (ug/ul)) = 1.5 ul pDNA into one tube
- Pipet 3 x 12 ul/ug = 36 ul Lipofectamine-2000 into the other tube
- Wait 5-10 min
- Transfer the content of the tube containing the pDNA into the tube containing the Lipofectamine-2000
- Incubate in the hood for 20 min
- Pipet dropwise evenly on the 10 cm diameter Petri dish containing the cells to be transfected
- Put the cells back in the 37°C/5% CO2 incubator
Day 3 : Transfer cells into 24-wells plate
Starting point: Petri dish with transfected cells
End point: 24-wells plate with transfected cells
- Dilute matrigel 100 x with cold DMEM (without any additives such as L-glutamine, FBS, pen/strep).
- Pipet 250 ul diluted matrigel into each well
- Incubate the plate for 30 min in the incubator
- In the meantime, wash and trypsinise the cells with 2 ml trypsin (6 cm dish). Centrifuge and resuspend in 13 ml DMEM (6 cm dish).
- Throw away the matrigel
- Pipet 1 ml cells into each well
Day 4 : Stimulate secretion
Starting point: 24-well plates containing attached transfected and untransfected GLUTag cells
End point: Frozen supernatants from stimulated cells
- Prepare washing solution: extracellular solution + 10 mM Glucose + 1 mg/ml BSA (Bovine serum albumin). E.g.: 50 ml extracellular solution + 90 mg Glucose (10 mM final concentration) + 50 mg BSA
- Prepare stimulating solutions:
A: 4 ml washing solution (negative control)
B: 4 ml washing solution + 4 ul forsklin at 10 mM (1000 X) + 4 ul IBMX at 100 mM (1000 X) (positive control)
C: 4 ml washing solution + 0.4 ul stimulating agent
D: 4 ml washing solution + 4 ul stimulating agent
- for (j in 1:3) {
for (i in 1:4) { # 4 rows in a 24-wells plate
empty row i
fill row i with 400 ul washing solution
}
}
- Empty row. Add 250 ul condition 3 wells at a time (the 3 wells containing the same condition)
- Incubate 2h at 37°C
- 30 min before the end of the incubation, prepare tubes. The total number of samples being N, we’ll need 2 x N tubes. The first set of tubes will be stored on wet ice (water ice) and the second set of tubes will be stored on dry ice (carbon dioxide ice). Both sets of tubes are labelled from 1 to N and the tubes in the second set are labelled with a barcode going from X to X + N – 1 (X being any integer).
- At the end of the incubation, transfer the plates from the incubator to wet ice and transfer the supernatants in the first set of tubes
- Spin the tubes for 5 min at 2000 rpm at 4°C
- Transfer the supernatant (be careful not to transfer any pellet) from the first set of tubes to the second set of tubes
- Store at -80°C until ready for analysis
Cristian Riccio, Laboratory of Prof. Gribble and Dr. Reimann, Institute of Metabolic Science
Activités scientifiques de Cristian Riccio