I've been trying to do the endocytosis pathway study of cell penetrating peptide under different pH value. So far, I've tried several different inhibitors including chlorpromazine, MBCD and Amiloride.
First, I pretreat HepG2 with the inhibitors for 30 min, then my cell penetrating peptide-EGFP fusion protein is added. Further incubation for 30 mins. Cells were washed with PBS twice and collected by using trypsin. And then cells were analyzed by using flow cytometry.
However!!! I don't get why the fluorescence of samples with the inhibitors (comparing to cell penetrating peptide-EGFP fusion protein only) was higher!!! like the fluorescence intensity was 150%~200%!
I really don't know what went wrong...
Are you stopping the experiment with ice-cold PBS? If so then does your trypsinisation take a long time or do you trypsinise warm? Its best if you trypsinise the cells and do the experiment while the cells are in suspension if you then need to facs them. Many of the endocytosis inhibitors are highly reversible so if your PBS or trypsin is warm then the increased PM bound ligand in the endocytosis blocked cells may then internalise as you wash your inhibitors off with PBS? If you are permeabilising the cells and counting total amounts and not surface levels then why not wash cells in ice-cold PBS, snap freeze and scrape to collect?
Hi Kelly
Do you know how your peptide work (the mechanism)? Could it be binding a receptor that suffers recycling? If this happens you may be actualy seing extracelular peptide that it's bound to cell surface (PBS may not be harsh enough to remove this kind of interaction). Could you perform your experiment using fluorescence microscopy or HCS? I believe it might be easier for you since you won't need to detach your cells.
Andre is right. Normally acid stripping or other such methods are used to remove ligand bound to the cell surface for endocytosis assays. There are numerous protocols available online and published.
Thank you all for answering!
I am washing my cells with PBS (Room temp). I tried both PBS and PBS with Heparin (20U/mL), and it seems like with either of these washing steps, fluorescence might still be higher than the control group.
Oh and yes... I do trypsinise my cells with warm trypsin solution (for 5 mins)
My peptide binds to surface proteoglycans before internalization. But I don't know about the further detail of its endocytosis mechanism...
Hi Kelly,
When I studied endocytosis, I used sodium azide to inhibit ATP. Initially, I pre-treated the cells with azide and then wash the azide away for CPP incubation. I did not get any inhibition. Later on, I co-incubate CPP with azide and I got no signal at all. So I am thinking the CPP internalization is very strong and need constantly inhibition. Maybe you can think about this approch.
Hi Kelly,
I read your question and I have a the same problem! I wonder about did you solve the problem? If you did how?
Hi Diğdem YÖYEN ERMİŞ
Unfortunately, I tired several washing conditions and even tried to not preheat the trypsin... nothing worked. Espeically with chlorpromazine!
Please do let me know if you find a way to solve this problem... haha
Hi Kelly,
Did you identify cell early , late apoptosis or dead cell when measuring flow cytometry? There is possibility cells are dying or dead so nonspecific uptake or binding increased a lot. I did some inhibition experiments and in my case, chlorpromazine caused serious cell death or cell morphology changed a lot with concentration people are using. Also, there is report chlorpromazine cause cell death for stressed cell over a large concentration range.
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