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Pore formation in cells after adding amphotericin B in culture medium

amphotericinb

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#1 S.C.

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Posted 26 January 2013 - 04:03 PM

Hello everyone,


I have some problem with amphotericin B. My lab had a bad time with fungal contamination. We had to discarded all culture and cleared the contamination for the several days :'(. After clearance, I decided to add 0.25 mg/ml amphotericin B (It's a working concentration which the company recommend.) in media. My medium is DMEM containing 10% FBS and 1% of penicillin/streptomycin. My culture is HepG2 which is hepatocyte.

BUT after changing I noticed many pore in my cells. I tried to find what happened with my culture but I could not find anybody discussed about this problem. I don't know that it was really the pore or it was the vesicles which happened because of the stress from the new condition of medium.

Now I remove this medium and add the medium without the amphotericin B.

I tried to find some information about amphotericin B. It is the antifungal reagent that commonly use in cell culture. It acts by binding to steroidal alcohols in cell membrane of susceptible fungi and creates channels in cell membrane resulting in an increase of membrane permeability. I also found some paper about AmB and hepatocytotoxicity (but I have not read in detail yet.).

There are my question
1. What should I do next? Am I doing right that I removed this medium and switched back to the normal one or I just caused more stress to my culture?
2. Can my culture recover back to normal? or what should I do to help them? or I have to thaw the new one? :'(


I do need some help. Anyone please help me.

Thank you for your kindness

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#2 bob1

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Posted 27 January 2013 - 12:39 AM

Those "pores" are called vacuoles and are a sign that the cells are stressed - this is pretty normal for cells treated with amphoterecin. Your best bit is to throw out these cells, clean everything (include changing your labcoat). These are a common cell line and are easily available so you should be able to get a non-contaminated stock.

For general cell culture, with appropriate sterile technique, you shouldn't need ANY antibiotics, let alone anti-fungals. The use of such agents damages the cells,causing them to behave in an abnormal manner and can hide low level contaminations that will also change how the cells behave.

#3 rhombus

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Posted 29 January 2013 - 06:14 AM

Dear S.C.

Using Amphotericin B is a very bad idea. Chuck away your current cells and decontaminate your class II cabinets/incubators/TC rooms where you can.

You have researched Amphotericin B and its mode of action and answered your own question. The vacuoles you see in your cells are the morphological evidence that the Amp B has altered cellular permability.

HOWEVER what you do not see is the changes in metabolism/biochemistry/pharmacology that have also occurred.

In the 1980's I was isolating endothelial cells from Porcine Thoracic Aorta. In the winter I got 80 % clean cells and by quarantining the cells I could isolate the infected ones and through them away. However in the summer the levels of both bacteria and fungus massively increased at the abattoir I visited. I could not find a way of getting a high percentage of clean cells. I did however start using Amp B as fungal contamination was my main problem. I got clean, unifected cells .............BUT the Nitric Oxide pathway that we were interested in disappeared i.e. unresponsive to NO agonist stimulation.

Thus the moral of the story is "if in doubt chuck them out (the cells)

Kindest regards

Uncle Rhombus




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