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How to retrieve my human blood immune cells from liquid nitrogen without killing


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3 replies to this topic

#1 uvbox

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Posted 25 August 2009 - 01:27 AM

Heya

I've stored some human peripheral blood immune cells in liquid nitrogen. Now i want to retrieve them as cell pellets so i can add trizol and extract RNA from these immune cells.

After i get them out of liquid nitrogen, i thawed them in water bath at 37 degrees, then i added them with RPMI medium into new vials.
As i was transferring them, i could see the cells were instantly forming debris (dying? didnt look good to me). I then spin them down at ~8000 xg for 5 mins, tipped out RPMI and treated cell pellets with trizol

After RNA extraction, i found my yield was really poor...

Anyone know a better way to do this?? or to avoid my cells dying as i mix them with RPMI ??

Any help is much appreciated!

#2 Clare

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Posted 25 August 2009 - 03:58 AM

You could try and culture the cells ON before you put them in TRIZOL - they will be happier that way. We do it with human blood cells. Question: why didn't you put some cells in trizol before liquid nitrogen?

Clare

Heya

I've stored some human peripheral blood immune cells in liquid nitrogen. Now i want to retrieve them as cell pellets so i can add trizol and extract RNA from these immune cells.

After i get them out of liquid nitrogen, i thawed them in water bath at 37 degrees, then i added them with RPMI medium into new vials.
As i was transferring them, i could see the cells were instantly forming debris (dying? didnt look good to me). I then spin them down at ~8000 xg for 5 mins, tipped out RPMI and treated cell pellets with trizol

After RNA extraction, i found my yield was really poor...

Anyone know a better way to do this?? or to avoid my cells dying as i mix them with RPMI ??

Any help is much appreciated!



#3 bob1

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Posted 25 August 2009 - 04:50 PM

How did you freeze them down? If you froze them just in medium, they are unlikely to survive. You will need to freeze them down in a mix of serum, medium and DMSO or other cryo-protectant.

#4 uvbox

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Posted 25 August 2009 - 04:59 PM

Hum...i think the cells were dying instantly as i added RPMI so im not sure if it would be any better if i culture them.
Yea, good idea didnt think of freezing them with trizol. Cool, will try that!

Cheers


You could try and culture the cells ON before you put them in TRIZOL - they will be happier that way. We do it with human blood cells. Question: why didn't you put some cells in trizol before liquid nitrogen?

Clare

Heya

I've stored some human peripheral blood immune cells in liquid nitrogen. Now i want to retrieve them as cell pellets so i can add trizol and extract RNA from these immune cells.

After i get them out of liquid nitrogen, i thawed them in water bath at 37 degrees, then i added them with RPMI medium into new vials.
As i was transferring them, i could see the cells were instantly forming debris (dying? didnt look good to me). I then spin them down at ~8000 xg for 5 mins, tipped out RPMI and treated cell pellets with trizol

After RNA extraction, i found my yield was really poor...

Anyone know a better way to do this?? or to avoid my cells dying as i mix them with RPMI ??

Any help is much appreciated!






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