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Intestinal Fibroblast culture - protocol


Best Answer bob1, 17 August 2014 - 04:06 PM

General contamination control is purely aseptic technique. Make sure you  keep your incubators, waterbaths, hoods etc clean, wear gloves, spray things with 70% ethanol before they go in the hood, don't touch surfaces when working etc.

 

Extraction contamination is more difficult. As you are (presumably) working with gut tissue, to extract the primary cells, you will need to make sure that you get rid of the bacteria that normally inhabit the gut. You might be able to do this through antibiotic treatment of the animals before you sacrifice them, but this probably won't get rid of all the bugs.

 

With the tissue, you should try first removing any fecal matter and as much of the gut mucus as possible (washing in Ringer's Lactate would probably work well for this), then bathe the tissue in a bacterial decontamination solution, such as 70% ethanol (iodine in ethanol might be better), then proceed with the extraction.  You will probably also need to add some antibiotics to the medium for the culturing as well.

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#1 Ahyi

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Posted 15 August 2014 - 12:48 AM

Hi Good Colleagues All,

 

Would you kindly suggest a good protocol that avoids contamination in primary intestinal fibroblast culture.

 

Thanks in advance.

 

A.



#2 bob1

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Posted 15 August 2014 - 02:55 PM

What sort of contamination - contamination from the extraction process? or just general contamination control?



#3 Ahyi

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Posted 17 August 2014 - 03:56 PM

Thanks. Both, from the extraction and general control.



#4 bob1

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Posted 17 August 2014 - 04:06 PM   Best Answer

General contamination control is purely aseptic technique. Make sure you  keep your incubators, waterbaths, hoods etc clean, wear gloves, spray things with 70% ethanol before they go in the hood, don't touch surfaces when working etc.

 

Extraction contamination is more difficult. As you are (presumably) working with gut tissue, to extract the primary cells, you will need to make sure that you get rid of the bacteria that normally inhabit the gut. You might be able to do this through antibiotic treatment of the animals before you sacrifice them, but this probably won't get rid of all the bugs.

 

With the tissue, you should try first removing any fecal matter and as much of the gut mucus as possible (washing in Ringer's Lactate would probably work well for this), then bathe the tissue in a bacterial decontamination solution, such as 70% ethanol (iodine in ethanol might be better), then proceed with the extraction.  You will probably also need to add some antibiotics to the medium for the culturing as well.



#5 Ahyi

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Posted 17 August 2014 - 04:46 PM

Did I say thank you? Thank you so much. Noted.

I use antibiotics in the medium at extraction and for culture. 


Edited by Ahyi, 17 August 2014 - 04:48 PM.





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