Hello,
We are examining a toxicant that increases the number of lipid droplets in THP-1 macrophages. We think this is due to agonism of cholesterol biosynthesis and subsequent storage in the droplet. I am trying to block the cholesterol biosynthesis pathway regulated by SREBP2 and bottlenecked by HMGCR with either Fatostatin (50uM) or Atorvastatin (25uM) which act on those targets, respectively. However, treatment with either drug alone significantly increases the MFI of BODIPY in the cells, the stain I am using to measure intracellular lipid content. Has anyone else encountered this and knows what causes it?
Thanks
To troubleshoot:
- Try lower drug concentrations
- Combine with an LDL receptor blocker
- Test in serum-free conditions
- Use radiolabeled acetate to confirm inhibition of de novo lipid synthesis
- Check expression of key lipid transporters (CD36, LDLR)
- Analyze the specific lipid species accumulating (cholesterol vs triglycerides)
Dear Braeden Giles I think I do not fully understand the question (some details are lacking to fully understand the point) but I guess it is a good thing to try to get in touch with https://www.researchgate.net/profile/Jason-Kilgore-2
He worked at Molecular Probes which is/are the inventor(s) of the BODIPY port folio. I think he can come up with some tips. You might check his answers to various questions as well, see for example https://www.researchgate.net/post/How_to_stain_lipid_droplets_using_bodipy
Best regards.
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