We are planning to determine specificity of SR-B1 protein in cholesterol uptake by using Dil-HDL. We managed to gather few references in order for us to develop a suitable methods. However the published method happened to incomplete and not reproducible. After few modification, we come out with these steps:
1. Seed HepG2 24-well plate (30000 cells/weel)
2. After 24 hours, treat the cells with desired treatment
3. After 24 hours treatment, wash the cells with PBS and incubate with 5 ug/ml of Dil-HDL at 37 degree celcius for 4 hours.
4. After incubation, wash the cells with PBS and lyse the cell with protein lysis buffer to obtain the desired protein. Transfer the cell lysate (protein) into a new plate and measure the absorbance at 520/ ext and 564 ems
5. The specificity Dil-HDL uptake is measure by carrying competitive assay in the presence of 40-fold excess of unlabelled HDL (this the part that I find hard to understand).
I also a bit confuse on how to dilute the Dil-HDL before using it since it comes in a liquid form (what solvent to use).
I hope we can spare our time to discuss this subject together.
Regards,
Luke
I haven't done DiI uptake assay in years and I never homogenized the cells. I took images instead, but I can tell you: Lipoproteins are stored in PBS containing 0.3 mM EDTA at 4°C in the dark to prevent oxidation. ( I made my own). I used CHO cells expressing (or not) the SR-BI receptor, not HepG2 cells, and it was important to grow the cells in lipoprotein-free medium 24h before and during the DiI-HDL incubation (I used 10 ug/mL). It was possible to detect nice DiI fluorescence as early as 10 min in the SR-BI expressing cells with a saturation at 5h. control cells also have a label, but significantly less. I don't remember doing a competition assay since I had a different control, but this is classic uptake protocol. Also, for imaging, you may want to check first the best time for your experiment. You don't want saturation. You want to see a label in the membrane and some intracellular dots. You might find this commercial kit protocol useful.
http://www.abcam.com/hdl-uptake-assay-kit-fluorometric-ab204717-protocols.html
Hi Soledad,
Thanks for your suggestion. We are now already purchased the kit and it s really helpful. Thanks!
Luke
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