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!
Thatīs 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!
Thatīs 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!
Thatīs 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!
Thatīs 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-