protein expression in NSO cells - (Jan/06/2008 )
I will use NSO cells to express some proteins in a large scale. But It seems that the cells are not adhesive well. So how can I get the stable cell line?
Is someone experienced in it? I need your help!
Many thanks!
-noirvan-
QUOTE (noirvan @ Jan 6 2008, 11:01 AM)
I will use NSO cells to express some proteins in a large scale. But It seems that the cells are not adhesive well. So how can I get the stable cell line?Is someone experienced in it? I need your help!
Many thanks!
That´s good, that they do not attach well as they are suspension cells....;-)
The easiest way of getting single cells would be the use of an FACS sorter after selection with the resistance marker (maybe even with a PI stain). If you do not have access to a sorter, you could also try to dilute the cells (best in 12 well plates) and then pick with a pipette under a micrsocope a single cell and put it into a 96-well U-shaped plate (it is better to have a U-bottom so that the cells after they divided are close to each other at the bottom of the well which increases cell survival).
Normally it is enough to pick 15-20 single cells when they have been already growing nicely under selection conditions. To ensure that you do not have by chance a mixture of cells (e.g. by accidentally picking more than one cell), you should repeat the picking with the colonies 2 to 3 times.
-Dukes-
I heard that NS0 is a good cell model for expressing exogenous proteins. There are three NS0 in ATCC. Which one I should buy? Can you share some experience with me ? Thanks. I should be able to help on stable cell line setup side.
QUOTE (Dukes @ Jan 7 2008, 05:52 AM)
QUOTE (noirvan @ Jan 6 2008, 11:01 AM)
I will use NSO cells to express some proteins in a large scale. But It seems that the cells are not adhesive well. So how can I get the stable cell line?Is someone experienced in it? I need your help!
Many thanks!
That´s good, that they do not attach well as they are suspension cells....;-)
The easiest way of getting single cells would be the use of an FACS sorter after selection with the resistance marker (maybe even with a PI stain). If you do not have access to a sorter, you could also try to dilute the cells (best in 12 well plates) and then pick with a pipette under a micrsocope a single cell and put it into a 96-well U-shaped plate (it is better to have a U-bottom so that the cells after they divided are close to each other at the bottom of the well which increases cell survival).
Normally it is enough to pick 15-20 single cells when they have been already growing nicely under selection conditions. To ensure that you do not have by chance a mixture of cells (e.g. by accidentally picking more than one cell), you should repeat the picking with the colonies 2 to 3 times.
-HCscientist-
QUOTE (HCscientist @ Feb 1 2008, 01:18 PM)
I heard that NS0 is a good cell model for expressing exogenous proteins. There are three NS0 in ATCC. Which one I should buy? Can you share some experience with me ? Thanks. I should be able to help on stable cell line setup side.
I will use NSO cells to express some proteins in a large scale. But It seems that the cells are not adhesive well. So how can I get the stable cell line?
Is someone experienced in it? I need your help!
Many thanks!
That´s good, that they do not attach well as they are suspension cells....;-)
The easiest way of getting single cells would be the use of an FACS sorter after selection with the resistance marker (maybe even with a PI stain). If you do not have access to a sorter, you could also try to dilute the cells (best in 12 well plates) and then pick with a pipette under a micrsocope a single cell and put it into a 96-well U-shaped plate (it is better to have a U-bottom so that the cells after they divided are close to each other at the bottom of the well which increases cell survival).
Normally it is enough to pick 15-20 single cells when they have been already growing nicely under selection conditions. To ensure that you do not have by chance a mixture of cells (e.g. by accidentally picking more than one cell), you should repeat the picking with the colonies 2 to 3 times.
QUOTE (Dukes @ Jan 7 2008, 05:52 AM)
QUOTE (noirvan @ Jan 6 2008, 11:01 AM)
I will use NSO cells to express some proteins in a large scale. But It seems that the cells are not adhesive well. So how can I get the stable cell line?Is someone experienced in it? I need your help!
Many thanks!
That´s good, that they do not attach well as they are suspension cells....;-)
The easiest way of getting single cells would be the use of an FACS sorter after selection with the resistance marker (maybe even with a PI stain). If you do not have access to a sorter, you could also try to dilute the cells (best in 12 well plates) and then pick with a pipette under a micrsocope a single cell and put it into a 96-well U-shaped plate (it is better to have a U-bottom so that the cells after they divided are close to each other at the bottom of the well which increases cell survival).
Normally it is enough to pick 15-20 single cells when they have been already growing nicely under selection conditions. To ensure that you do not have by chance a mixture of cells (e.g. by accidentally picking more than one cell), you should repeat the picking with the colonies 2 to 3 times.
Where do you find NS0 cells at ATCC? The three hits with a "NS0" search are all hybridoma cells which were generated by fusion with NS0 cells (CRL-1824, 2695,2696).
-Dukes-
You are right. Where can I find original NS0 cells? Your help is much appreciated.
QUOTE
Where do you find NS0 cells at ATCC? The three hits with a "NS0" search are all hybridoma cells which were generated by fusion with NS0 cells (CRL-1824, 2695,2696).
-HCscientist-
QUOTE (HCscientist @ Feb 6 2008, 12:13 AM)
You are right. Where can I find original NS0 cells? Your help is much appreciated.
QUOTE
Where do you find NS0 cells at ATCC? The three hits with a "NS0" search are all hybridoma cells which were generated by fusion with NS0 cells (CRL-1824, 2695,2696).
at the European Collection of Cell Cultures
http://www.ecacc.org.uk/
-Dukes-
I have a question:
It is well known that CHO cells are used for expressing many proteins.
So what's the difference between CHO and NS0 for recombinant protein expression? yield or something else?
or what's the advantage of NS0?
did anyone has experience of this kind?
first time to post , Many thanks!! 
-davidchang-