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Created page with '==Materials== �*Cells in 100 mm culture dishes �*Labelling Media (Inositol Free DMEM with PSG, 10% dialysed serum and 100 uCi inositol per plate of cells) �*Perchloric acid...'
==Materials==
�*Cells in 100 mm culture dishes
�*Labelling Media (Inositol Free DMEM with PSG, 10% dialysed serum and 100 uCi inositol per plate
of cells)
�*Perchloric acid (640 uL into 10 mL water) keep cold
�*100 mM EDTA (2 mL of 0.5M stock in 10 mL) keep cold
�*Water
�*Deacylation Mix (3.5 mL Methylamine, 6 mL Methanol, 1.5 mL Butanol, 2.1 mL Water)
�*Lipid Extraction Mix (10 mL Butanol, 2 mL Ethyl Ether and 0.5 mL Ethyl Formate)
==Protocol==
#Label subconfluent cells (60-80% confluence) for 48h in labelling media. If using adipocytes label at 4-6 days post-FBS for 48h
#Check that eppendorf centrifuge is at 4C and that heating block (with right size holes) is at 56C
#Treat cells as required(remove media if necessary)
#Aspirate media and add 1 mL Perchloric acid. Incubate at 4 C for 15 min
#Scrape cells, transfer to eppendorf tubes and spin 10 min at 16 000g at 4 C
#Aspirate supernantant and resuspend cells in 1 mL 100 mM EDTA. Centrifuge again.
#Suspend pellet in 50 uL of water.
#Deacylation
##Add 1 mL Deacylation mix and transfer to glass tube
##Incubate at 56 C for 45 min
##Dry under vacuum
#Resuspend pellet in 0.5 mL water. Vortex 30s
#Centrifuge at room temperature for 2 min
#Extract Lipids:
##Remove supernatant to tube with 0.5 mL Lipid Extraction Mix
##Vortex 15s
##Centrifuge at room temperature for 2 min
##Remove lower phase and repeat steps a-c once
#Remove lower phase to clean tube and dry under vacuum.
#Store dried samples at -20 C
==References==
[1] C C Whiteford, C Best, A Kazlauskas, and E T Ulug. D-3 phosphoinositide metabolism in cells treated
with platelet-derived growth factor. Biochem J, 319 ( Pt 3):851{60, Nov 1996.
�*Cells in 100 mm culture dishes
�*Labelling Media (Inositol Free DMEM with PSG, 10% dialysed serum and 100 uCi inositol per plate
of cells)
�*Perchloric acid (640 uL into 10 mL water) keep cold
�*100 mM EDTA (2 mL of 0.5M stock in 10 mL) keep cold
�*Water
�*Deacylation Mix (3.5 mL Methylamine, 6 mL Methanol, 1.5 mL Butanol, 2.1 mL Water)
�*Lipid Extraction Mix (10 mL Butanol, 2 mL Ethyl Ether and 0.5 mL Ethyl Formate)
==Protocol==
#Label subconfluent cells (60-80% confluence) for 48h in labelling media. If using adipocytes label at 4-6 days post-FBS for 48h
#Check that eppendorf centrifuge is at 4C and that heating block (with right size holes) is at 56C
#Treat cells as required(remove media if necessary)
#Aspirate media and add 1 mL Perchloric acid. Incubate at 4 C for 15 min
#Scrape cells, transfer to eppendorf tubes and spin 10 min at 16 000g at 4 C
#Aspirate supernantant and resuspend cells in 1 mL 100 mM EDTA. Centrifuge again.
#Suspend pellet in 50 uL of water.
#Deacylation
##Add 1 mL Deacylation mix and transfer to glass tube
##Incubate at 56 C for 45 min
##Dry under vacuum
#Resuspend pellet in 0.5 mL water. Vortex 30s
#Centrifuge at room temperature for 2 min
#Extract Lipids:
##Remove supernatant to tube with 0.5 mL Lipid Extraction Mix
##Vortex 15s
##Centrifuge at room temperature for 2 min
##Remove lower phase and repeat steps a-c once
#Remove lower phase to clean tube and dry under vacuum.
#Store dried samples at -20 C
==References==
[1] C C Whiteford, C Best, A Kazlauskas, and E T Ulug. D-3 phosphoinositide metabolism in cells treated
with platelet-derived growth factor. Biochem J, 319 ( Pt 3):851{60, Nov 1996.