Hi all - I am interested to hear from experts in LDL uptake assays. We use BODIPY-LDL from Molecular Probes and LPDS from Sigma or Millipore with unmodified HepG2 cells to measure LDL uptake. Plates are Poly-D-Lysine from Greiner. We see high well-to-well variability between identical replicates in LDL uptake leading to assay failure. What can be the reason for this? We suspect LPDS lot and plate lot issues. Briefly:
Day 1: Plate 20K/well HepG2 cells in DMEM-LPDS media on Greiner Poly-D-Lysine 96-well plates. Incubate overnight.
Day 2: Add drug modifying LDL uptake, incubate 4 hours. Add BODIPY-LDL. Incubate overnight.
Day 3: Wash wells with PBS, add PBS to avoid cell drying, and read uptake on fluorimeter.
All suggestions would be appreciated!
Hi Anton,
I have no experience with BODIPY-lablelled LDL, but I used Dil-LDL for cellular uptake assays. I realized that the right concentration was very important, as with too much signal, I also got uneven values between wells and experiments. I am not sure, if you really need ON LDL uptake for your readout. Liver cells should be quite active in LDL uptake. In my cell culture assay I got already a good signal after 2h. Additionally, it might be that your LDL/BODIPY sticks to the plastics. I used cells grown on coated glass coverslips, what worked quite well for me. What is labelled in your LDL? Is it apoB or some lipid component? It might also be, that your label was removed from the particle. I heard that SRBI on the cell surface can at least remove Dil-label from LDL. Maybe you can look at a colocalisation with LDLR to make sure that your label stays with the LDL.
I hope the information will help you and good luck with your experiments.
Hi Anton,
some first thoughts about the assay.
- Why do you use poly-D-lysine plates? HepG2 should stick quite well in regular cell culture plates.
- Is an overnight incubation with BODIPY-LDL required? Is a couple of hours not enough?
- Is fluorescence of BODIPY higher in organic solvents to extract it from the cells instead of just leaving PBS on the washed cells?
- If you add unlabelled LDL to the BODIPY-LDL, is you fluorescent signal reduced after uptake?
- Providing some more details on the assay-protocol might help solving your issue.
Regards,
Mario
Dear Anton
You are not vortexing the LDL are you? Vortexing causes aggregation of LDL, which would increase its uptake.
Regards
David
We did not work with HepG2 cells so far but on the uptake of LDL by macrophages in cell culture: The chapter materials and methods of a paper by us may help you: " Binding and uptake of differently oxidized low density lipoprotein in mouse peritoneal macrophages and THP-1 macrophages: Involvement of negative charges as well as oxidation-specific epitopes
Author(s): Wang, XS; Greilberger, J; Ledinski, G; et al.
Source: Journal of Cellular Biochemistry Volume: 81 Issue: 3 Pages: 557-569 Published: 2001"
Regards
Günther
Wow thank you guys! So many great ideas. Unfortunately, I couldn't change too many things because we use the assay in the regulated drug manufacture QC environment. But I will definitely act on some points:
1. Minimize LDL uptake time within the scope of the protocol.
2. Christine, what is the optimal concentration of LDL you have used? We aim to use 0.01 mg/ml of BODIPY-LDL. Should we optimize it empirically? Any references on the topic?
3. Christine, BODIPY-LDL is quite similar to DiI-LDL, just a different dye with 20X quantum yield for better signal. It's not covalently attached to protein or lipid. It's quite possible that it can be removed from LDL under certain conditions. I will think about ways to ensure that BODIPY-LDL stays intact.
4. Mario, we found that regular plates don't withstand washing on the last day of the assay to remove excess of labeled LDL. In fact, cell loss even with the Poly-D-Lysine plates is a major headache with this assay.
5. Mario, excellent suggestion regarding extraction of BODIPY-LDL from the cells at the end of the protocol. If you have a protocol, I would appreciate it very much and will try to test it.
6. Mario, you are absolutely right: we have found that inferior lots of LDL-depleted serum cause assay failure. We are moving towards qualifying LPDS lots to ensure we have minimal unlabeled LDL levels in our assay media.
7. David - very good point: we will try to avoid any excessive vortexing from now on.
8. Guenther, thanks for the reference. I will read it carefully. I wonder if unmodified HepG2 cells have a heterogeneity of LDL receptor on the surface. Or if LDL receptor is regulated in some way to cause differences in well-to-well uptake. In any case, thanks for all the ideas, they are very helpful and actionable. Anton
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