Sackler School of
Graduate Biomedical Sciences

Transport of LDL-derived [³H]cholesterol to the plasma membrane as measured by accessibility to cyclodextrin

Seed cells in 6-well plates. After 24 hr, refeed cells medium with LPDS. After 48 hr, make staggered addtions of [³H]CO-LDL. Wash cells twice with PBS (chilled to 4^oC). Add 1 ml 12.5 mM 2-hydroxy-propyl-b-cyclodextrin (Sigma) (CD) in serum-free medium. Incubate cells 10 min at 37^oC.

Media –
Pipet media into microfuge tubes. Spin at 4^oC 2 min to pellet any cells. Transfer media to 13X100 glass test tubes containing 1 ml ethanol and 10 ul rLDL recovery standard. Vortex. Store at 4^oC until ready to extract.

Vortex media samples. Extract twicewith 3 ml petroleum ether. Pool PE extracts (top layer) in 13X100 mm glass tubes. Backwash (add1 ml water, vortex, discard lower layer using long Pasteur pipet). Dry PE. TLC with toluene/ethyl acetate (2:1) using 20X20 plastic-backed silica gel G TLC plates that are cut in half. Spot 10 ul of recovery standard on both ends of plate (cold rLDL). Cut out iodine sensitive spots corresponding to cholesteryl ester and cholesterol.

Cells –
Dishes with cells are brought into cold room and washed 1x quickly, 2x 7 min (on rotator) with TBS-BSA (2 mg/ml), then twice quickly with TBS. Aspirate TBS carefully. Extract cell lipids with hexane/isopropanol (3:2). Add 2 ml hex/IP to each well and incubate for 15 min on rotator in cold room. Bring plates into lab and add 10 ul rLDL recovery standard. Place on lab rotator for 15 min. Transfer extracts into 12X75 tubes. Rinse wells with 1 ml hex/IP, pool extracts. Store on lab bench until ready to TLC

Dry hex:IP extracts. TLC with toluene/ethyl acetate (2:1) using half TLC plates as above.
Cut out iodine sensitive spots corresponding to cholesteryl ester and cholesterol.

Protein per well –
Let dishes air dry; add 1 ml 0.1 N NaOH and swirl 1 hour at RT. Transfer 200 ul aliquots to 12X75 tubes. Set up BSA standard tubes with 0, 5, 10, 20 ul 1 mg/ml BSA and 200 ul 0f 0.1 N NaOH. Add 1 ml Lowry reagent, vortex, wait 10 min. Add 50 ul Folin:phenol, vortex, wait 30 min. Read OD 670 nm.

rLDL rec std = 2000 dpm [¹⁴C]cholesterol, 40 ug cholesterol, 20 ug cholesteryl oleate per 10 ul of chloroform.

Reference: Wojtanik and Liscum (2003)
Modified September 2006