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Thawing and Plating


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4 replies to this topic

#1 borriello.87

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Posted 02 September 2009 - 10:43 PM

Hi,
I'd like to ask you some questions about what to do after thawing lymphocytes.
do I have to plate thawed lymphocytes before extracting RNA or performing a flow cytometry analysis? If yes, do I have to use medium with or without serum?

Thanks in advance.

#2 Stephan

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Posted 02 September 2009 - 10:55 PM

I'm not sure how long it would take for the cells to resume their natural morphology (after dehydration). I would at least leave for a couple of hours in media with serum.

Flow cytometry would need this step but RNA extraction I think would not matter.

#3 molgen

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Posted 03 September 2009 - 01:47 AM

For RNA extractions you don't need to plate the cells. Pellet them and do the extraction.
The same for flow cytometry.

#4 bob1

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Posted 06 September 2009 - 05:14 PM

It depends on what you are looking at - some RNAs are expressed very quickly in response to stresses (e.g HSP70 in response to heat shock) so ideally you would get the cells in the most "normal" state you can so as to not bias your results. Personally I would seed them out and wait for a couple of days to allow cell membrane components to recover and normal levels of growth genes etc to be established.

#5 borriello.87

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Posted 08 September 2009 - 03:06 PM

Thank you for all your answers, they've been very useful!!!




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