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CLOUDINESS of RIN5F cell culture


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#1 sree99

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Posted 19 November 2012 - 10:09 PM

Hello

I have cultured RIN5F- cell line in RPMI supplemented with penstrep,LGlu and Amp-B..subcultured and freezed some cells in 10%DMSO.
Recently I revived some cells and after 4-5 days , I noticed the flask is bit cloudy and I can clearly see some kind of flocculation.

Is it the contamination?
or
the rin5f cells secrete some peptide???

Can I plate the cells now for experiment or discard it?

n

one more


Do I got to prepare the RPMI/+ constituents freshly every time or I can keep filtered stock of 200ml??
(cos i used the media from stock?)

Please do send me your sggestions

Sree99

#2 bob1

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Posted 20 November 2012 - 01:03 AM

Probably bacterial or yeast contamination. General rule is "If in doubt, throw them out" - if you are worried now - how will using them affect your experiments...?

Could just be a lot of dead cells - have a look down the microscope - yeast show small round cells often with budding, bacteria will appear as a shimmer in the background and dead cells will look like dead cells and debris.

#3 sree99

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Posted 20 November 2012 - 09:07 PM

Thanks for your response.
Yesterday ..though changed the media..for the final try...today still I see some kind of precipitation in flask..n cells too seem to be detached.
Today am goin to revive the RINcells

Any suggestions for my revival???

cos
some say revived contents+10ml culture media and then centrifuge @200g for 5min
to the pellet, add again culture media n then transfered to t25

n
some..directly pouring contents into t25

please..this time I got to save my cells ...n start my expt..Posted Image

#4 bob1

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Posted 21 November 2012 - 12:38 PM

It depends on the cells, usually it is best to remove the freezing medium as it contains DMSO, which is quite toxic. For this you would do your first protocol above, making sure that you remove the medium after pelleting the cells. Transfer to an appropriate sized flask or well depending on the viability of the cells and the amount frozen down.

I would also suggest that you get someone experienced to check on your cell culture techniques - if you are getting contamination with your cocktail of antibiotics you are doing something seriously wrong... if your technique is correct, you shouldn't need antibiotics at all, and certainly not antifungals such as amphotericin, which are also toxic to your cells!

#5 sree99

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Posted 21 November 2012 - 09:21 PM

It depends on the cells, usually it is best to remove the freezing medium as it contains DMSO, which is quite toxic. For this you would do your first protocol above, making sure that you remove the medium after pelleting the cells. Transfer to an appropriate sized flask or well depending on the viability of the cells and the amount frozen down.

I would also suggest that you get someone experienced to check on your cell culture techniques - if you are getting contamination with your cocktail of antibiotics you are doing something seriously wrong... if your technique is correct, you shouldn't need antibiotics at all, and certainly not antifungals such as amphotericin, which are also toxic to your cells!


MANY thanks for your response.
While reviving...if the media eg RPMI suggested for cell line require 123456..
n
we have only 123
is that ok for revival??

Currently I dont have HEPES n sodium pyruvate..n my sup..ordered..it!!
probably i get around dec 2nd week.

I attach pic of contaminated culture. what are all those background spots???......
juss.........

How do I attach pics here???

#6 bob1

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Posted 22 November 2012 - 11:49 AM

The HEPES is used for buffering of the solution to keep it at physiological pH. Pyruvate is an energy source for the cell... you need these things! Don't thaw the cells without them.

Images can be attached using the full reply option (extra reply option next to the quick reply button). Only some image formats are accepted. I think .bmp and .jpg should work.

#7 sree99

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Posted 27 November 2012 - 09:06 PM

The HEPES is used for buffering of the solution to keep it at physiological pH. Pyruvate is an energy source for the cell... you need these things! Don't thaw the cells without them.

Images can be attached using the full reply option (extra reply option next to the quick reply button). Only some image formats are accepted. I think .bmp and .jpg should work.


Oh K .
Thanks
n One more..which is the best for rin5f cell culture?

HEPES or sodium salt of HEPES?
I would like to know difference between two in terms of good buffer?!

#8 bob1

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Posted 28 November 2012 - 12:52 AM

The difference is Na (sodium) which adds to the ionic content of the medium, depending on how much you are adding this could be a problem. I would go for the plain HEPES.




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