Differentiation of MEFs: Difference between revisions

Line 24:

# Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).

# On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).

3. At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off).

# At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off). '''Note1:''' Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)

• Note1 Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)

# Protein lysate and RNA extraction is taken at day 4 (D3). '''Note2:''' In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection. '''Note3:''' A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

4. Protein lysate and RNA extraction is taken at day 4 (D3).

• Note2: In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection.

• Note3: A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

@@ Line 24: / Line 24: @@
 # Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
 # On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
-.	At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off).
+# At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off). '''Note1:''' Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
-•	Note1 Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
+# Protein lysate and RNA extraction is taken at day 4 (D3).  '''Note2:''' In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection.	'''Note3:''' A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.
-.	Protein lysate and RNA extraction is taken at day 4 (D3).
-•	Note2: In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection.
-•	Note3: A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.