RAW264.7 cells+nitric oxide determination - (Jul/26/2009 )
Dear Rhombus uncle,
Can you please let me know whats the minimal time for nitric oxide production in j774 macrophages when incubated in presence of LPS(in mg). I was wondering how long does a macrophage take to produce nitric oxide. Can you please also let me know where can I get these basic information from? I did enough review, but failed to find any answer.
Hope you would be able to help me out.
In anticipation of your reply,
Regards,
Nethra
http://onlinelibrary.wiley.com/doi/10.1080/15216549600201503/pdf
Dear Nethra,
Please look at the above paper which has a perfect time course for iNos induction.
Basically if you do a western to look at protein expression, iNOS protein will be present from 4 hours post addition of LPS. However you will not yet be able to measure Nitrite in the cell culture media. Our experiments agree with the time course graph in the above paper i.e. you will start measuring nitrite at 8 hours. Maximum levels will probably be achieved at 24-36 hours post induction. By this time the level of NO produced starts to kill the cells.
Hope this is useful.
Kindest regards.
Uncle Rhombus.
I know I am off topic. But since Uncle Rhombus is here, I hope he would come across my question.
Hi, I didnt know that cells could not be kept above -70. I kept it at -50 to -30 for 2 weeks (because the freezer in my lab cannot decrease the temperature anymore. It just maintain from -30 to -50). After I realized it, I transferred it to liquid nitrogen. Can my cells still be revived? What are the chances? And how can I increase the viability?
By the way, the cell is RAW cells. And I havent thawed it since receiving it from ATCC.
P/S: I am new to cell culture.
citomebo on Thu Feb 16 03:18:53 2012 said:
I know I am off topic. But since Uncle Rhombus is here, I hope he would come across my question.
Hi, I didnt know that cells could not be kept above -70. I kept it at -50 to -30 for 2 weeks (because the freezer in my lab cannot decrease the temperature anymore. It just maintain from -30 to -50). After I realized it, I transferred it to liquid nitrogen. Can my cells still be revived? What are the chances? And how can I increase the viability?
By the way, the cell is RAW cells. And I havent thawed it since receiving it from ATCC.
P/S: I am new to cell culture.
Dear citomebo,
The standard protocol for cell freezing is that the cells are cooled and frozen slowly in a controlled rate freezing machine at 1oC/minute. Researchers nowadays use "Mr. Frosties" which are very cheap and freeze at similar rates. Both methods allow cells to be frozen down to -80oC. The cells are then stored in liquid nitrogen for long periods....I have cells that have been frozen for 25 years and still are easily recovered.
To answer you specifically:
"Can they be revived".........you will have to check them yourself.
"What are the chances"......my guess is that RAW cells are very difficult to kill, so I would imagine that some cells will recover, but your viability may be reduced significantly. Also you are putting a "selection pressure" on these cells i.e. only the cells that can withstand this insult will survive. In all cell lines there are varying clones growing and you may select one clone over another.
"And how can I increase viability"........Cells are only used as a model to test a hypothesis. I do not know what you intend to do with these RAW cells. If it was me in your position I would purchase new cells. However money may be tight so why don't you get them growing (if they will) and then stimulate them with something like LPS...and test if they are producing Nitric oxide for example.....then you know that
a) they are viable
they are metabolically active.Hope this helps in some way.
Kindest regards
Uncle Rhombus