Hi everyone,
I have been using this technical note from BMG Labtech (https://www.bmglabtech.com/en/application-notes/membrane-fluidity-measurement-using-uv-fluorescence-polarization/) and trying to establish an assay to measure the fluidity of various liposomes (Small Unilamellar Vesicles ~50-100 nm). My liposomes have POPC and other lipids like Cholesterol and Sphingomyelin at different mol%. I have tried labeling the liposomes with 2.5-10 uM DPH based on what I saw in the literature (from a 1mM stock in DMSO) at 45°C for 30 minutes and then took endpoint FP measurements over a temp range of 25-45°C. However, in most experiments, I see a gradual increase in FP value as temp increases which indicates the fluidity is decreasing. To my understanding, the fluidity is expected to increase with an increase in temp. Adding Triton to these DPH-labelled liposomes, however, result in a sharp decrease in FP, indicating the disruption of the liposomes.
Can anyone please share their experiences with this assay and any possible reason why I am seeing an increase in FP as the temp increases? I have searched quite a lot of literature but seem not to find how the specific lipid combination I am using should influence liposome fluidity with increasing temp. Should I try labeling the liposomes at room temperature instead? I will greatly appreciate any leads!
#membranefluidity #DPH #fluorescencepolarization #anisotropy
Dear Antara Chakravarty ,
It could be caused by numerous different reasons I’m afraid. For example, it is known that cholesterol can cause a deeper insertion of the DPH in the membrane and as such DPH fluorescence intensity changes are not always indicative for changes in membrane fluidity. But trying to look for a simple explanation first:
-Did you try a POPC membrane as well and were you able to repeat findings in the literature?
-What ratio of DPH:Lipid did you used? The literature I know use ratios like 1:300 or 1:250.
-Not sure whether you (and the literature you refer to) talk about 10 uM as end concentration but this should be (at least in ‘normal’ fluorescence measurements) in the range of 0.1-1.0 uM.
I ask this because it sounds to me that while measuring at different temperatures there seems to be still numerous DPH molecules that are getting embedded (not labeled…) in the lipid membrane. Since DPH is essentially non-fluorescent in water, the increase might be explained by the DPH that still gets incorporated in the membrane. I think that when you start not all DPH is incorporated in the membrane. You could try ethanol instead of DMSO (at lower concentrations) since ethanol is better miscible with water to see whether more DPH gets incorporated in the membrane.
Best regards.
PS. According to some of the literature you read as well, it is common when you look at different temperatures to wait for quite a while (something like 30 minutes or so) before the ‘endpoint’ is reached.
Best regards.
Rob Keller Thank you so much, Dr. Keller! Keeping all your suggestions in mind, I am repeating the experiment this week, and I will let you know the results. Thanks!
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