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:

End point:

pDNA stock concentration: 2 ug/ul

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

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

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