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

1. Transfer 5 ml of condition medium into a falcon tube.
2. Throw away the rest of the condition medium (should be 15 ml)
3. Wash cells with 10 ml PBS
4. 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)
5. During the trypsinisation, add 2 ml complete DMEM to the 5 ml condition medium
6. At the end of the trypsinisation, add the 7 ml medium and triturate the cells. The total volume of cells is now 10 ml
7. Add 6 ml complete DMEM to each of two 10 cm diameter Petri dishes
8. Add 4 ml of resupspended cells to each of the Petri dishes
9. Add the remaining 2 ml of resuspended cells to a new T75 flask containing 18 ml complete DMEM
10. 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)

1. Pipet 750 ul of Optimem in each of two eppendorf tubes
2. Pipet 3 ug/(2 (ug/ul)) = 1.5 ul pDNA into one tube
3. Pipet 3 x 12 ul/ug = 36 ul Lipofectamine-2000 into the other tube
4. Wait 5-10 min
5. Transfer the content of the tube containing the pDNA into the tube containing the Lipofectamine-2000
6. Incubate in the hood for 20 min
7. Pipet dropwise evenly on the 10 cm diameter Petri dish containing the cells to be transfected
8. 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

1. Dilute matrigel 100 x with cold DMEM (without any additives such as L-glutamine, FBS, pen/strep).
2. Pipet 250 ul diluted matrigel into each well
3. Incubate the plate for 30 min in the incubator
4. 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).
5. Throw away the matrigel
6. 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

1. 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
2. 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
3. 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
   }
   }
4. Empty row. Add 250 ul condition 3 wells at a time (the 3 wells containing the same condition)
5. Incubate 2h at 37°C
6. 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).
7. 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
8. Spin the tubes for 5 min at 2000 rpm at 4°C
9. Transfer the supernatant (be careful not to transfer any pellet) from the first set of tubes to the second set of tubes
10. Store at -80°C until ready for analysis

Cristian Riccio, Laboratory of Prof. Gribble and Dr. Reimann, Institute of Metabolic Science