Mbs173147

Cholesterol efflux was determined using the cholesterol efflux fluorometric Assay Kit (Cat. K582100; BioVision, Milpitas, CA, USA). Briefly, macrophages were plated on 96-well plates (1X10⁵ /well) and labeled with fluorescently-labeled cholesterol analogue for 1 h. After equilibration in culture for 16 h, cells were incubated without (negative control) or with 10 mM methyl-β cyclodextrin (positive control) or 50 μg/ml human HDL(MBS173147, MyBioSource, San Diego, CA, USA) as indicated in phenol red-free, serum-free RPMI medium for 6 h. The culture medium and cell lysate were separately collected and the fluorescence intensity (RFU) was determined using a microplate reader (SpectraMax Gemini EM Microplate Reader, Molecular Devices, San Jose, CA, USA). The percentage of cholesterol efflux = [RFU of medium/(RFU of cell lysate + RFU of medium)] × 100.

For efflux, Huh7 or HT22 cells were incubated with 2 μM C6-NBD Cer in DMEM with 10% FBS at 37 °C for 3 h and washed three times with DMEM plus 0.1% BSA. Efflux was initiated by the addition of fresh DMEM containing 40 μg/ml of either BSA or human serum HDL (#MBS173147, MyBioSource). In some experiments, Huh7 cells were also incubated with 0.2 μM [¹⁴C]-Cer, instead of C6-NBD Cer, to study sphingolipid synthesis and efflux. Culture media were harvested after 1 h or 6 h of incubation and centrifuged (2500 rpm, 600g, 15 min, 4 °C, Heraeus Fresco 21 Centrifuge, rotor 75003424, Thermo Fisher Scientific) to pellet detached cells. Lipids in the media and cells were extracted (43 (link)), dried, and resuspended in 100 μl of isopropanol for the separation of sphingolipids on TLC silica plates (#44931, Analtech, Inc) with a CHCl₃:CH₃OH:C₆H₅CH₃:NH₄OH:H₂O (40:40:20:0.4:1.6, ratio by volume) solvent system (4 (link), 5 (link)). The TLC plates were visualized with a PhosphorImager (GE Healthcare), and bands corresponding to Cer, GluCer, and SM were quantified in ImageJ software.