Acetate Incorporation into Lipid: Difference between revisions

(One intermediate revision by the same user not shown)

Line 7:

* Cells plated in 12 well dishes

* Low Glucose Starvation Media (5 mM Glucose, 0.5% FBS)

* Acetate Incorporation Media (Low Glucose Starvation Media + 2 mM Acetate and ~~0.1~~ uCi/mL 14C Acetic Acid)

* Acetate Incorporation Media (Low Glucose Starvation Media + 2 mM Acetate and 10 uCi/mL 14C Acetic Acid)

* PBS at 4C

* [2-14C] Acetic Acid. Perkin Elmer Cat# NEC085H001MC

Line 15:

# Turn radioactive tissue culture incubator on at 37C, make sure to turn on the CO2 as well.

# Starve cells in '''Low Glucose Starvation Media''' for >3h.

# Prepare '''Acetate Incorporation Media''' Make at least enough for one extra well, using ~~0.5 mL~~/well for a 12 well dish. If monitoring lipogenesis in a line other than adipocytes, increase the hot glucose to ~~0.5~~ uCi per mL.

# Prepare '''Acetate Incorporation Media''' Make at least enough for one extra well, using 50 uL/well for a 12 well dish. If monitoring lipogenesis in a line other than adipocytes, increase the hot glucose to 50 uCi per mL.

# Pretreat cells with inhibitors if required.

# ~~Change Media to '''Acetate Incorporation Media''' and add~~ 100 nM insulin as required.

# Add 100 nM insulin as required.

# Place in the radioactive tissue culture incubator.

# Add 50 uL of Hot Acetate Solution to each well.

# Place in the radioactive tissue culture incubator for 60-120 minutes.

# Save 3 x 5 uL of the '''Acetate Incorporation Media''' to count total radioactivity.

# After 60 min wash cells 3x with 1 mL of PBS.

@@ Line 7: / Line 7: @@
 * Cells plated in 12 well dishes
 * Low Glucose Starvation Media (5 mM Glucose, 0.5% FBS)
-* Acetate Incorporation Media (Low Glucose Starvation Media + 2 mM Acetate and 0.1 uCi/mL 14C Acetic Acid)
+* Acetate Incorporation Media (Low Glucose Starvation Media + 2 mM Acetate and 10 uCi/mL 14C Acetic Acid)
 * PBS at 4C
 * [2-14C] Acetic Acid.  Perkin Elmer Cat# NEC085H001MC
@@ Line 15: / Line 15: @@
 # Turn radioactive tissue culture incubator on at 37C, make sure to turn on the CO2 as well.
 # Starve cells in '''Low Glucose Starvation Media''' for >3h.
-# Prepare '''Acetate Incorporation Media''' Make at least enough for one extra well, using 0.5 mL/well for a 12 well dish.  If monitoring lipogenesis in a line other than adipocytes, increase the hot glucose to 0.5 uCi per mL.
+# Prepare '''Acetate Incorporation Media''' Make at least enough for one extra well, using 50 uL/well for a 12 well dish.  If monitoring lipogenesis in a line other than adipocytes, increase the hot glucose to 50 uCi per mL.
 # Pretreat cells with inhibitors if required.
-# Change Media to '''Acetate Incorporation Media''' and add 100 nM insulin as required.
+# Add 100 nM insulin as required.
-# Place in the radioactive tissue culture incubator.
+# Add 50 uL of Hot Acetate Solution to each well.
+# Place in the radioactive tissue culture incubator for 60-120 minutes.
 # Save 3 x 5 uL of the '''Acetate Incorporation Media''' to count total radioactivity.
 # After 60 min  wash cells 3x with 1 mL of PBS.