Hi Raghunath,

                    Zebrafish normally grow at 26-28.5 deg. C. If you grow zf cells at 37 deg C, they would undergo acute stress, upregulate heat shock genes and die over time. We use 37-42 deg C for activating heat shock promoters and fish do not like this. You may be able to grow zf cells at room temperature if your room temperature is stable. Look for media that does not require CO2. BTW none of the above papers say anything about zf cell lines growing at 37 deg C

Good luck...

Dear Raghunath Azhwar, 

The attached three publications depict some zebrafish cell lines grow at 37 degrees such as AB-9 and etc.

Hoping this will be helpful,

Rafik

Thank you for your time, Sir. 

Hi Raghunath,

                    Zebrafish normally grow at 26-28.5 deg. C. If you grow zf cells at 37 deg C, they would undergo acute stress, upregulate heat shock genes and die over time. We use 37-42 deg C for activating heat shock promoters and fish do not like this. You may be able to grow zf cells at room temperature if your room temperature is stable. Look for media that does not require CO2. BTW none of the above papers say anything about zf cell lines growing at 37 deg C

Good luck...

Dear Sandeep,

As I understood from the question, the incubation has to be at 37 C. Therefore, I did not mention temperatures outside 37 C. Although the three publications attached to my previous answer described the incubation at 28 C and the effect of CO2 on the incubation.

Regarding your answer that the papers did not point out the use of 37 C, I have copied a paragraph from the first review article which states:

"Mammalian cells require 34– 37 C, whereas fish cells can be grown over a wide temperature range. For example, rainbow trout cells can be grown at temperatures between 5 and 26 C, but even at optimal temperatures (18– 22 C), they grow more slowly than mammalian cells at 37 C".

This paragraph clearly states that fish cells can grow at 37 C but in slower rate compared to temperatures of 18-22 C.

Hoping I clarified my point.

Rafik

Hi Rafik,

        The paragraph you mentions says

" Mammalian cells require 34–37°C, whereas fish cells can be grown over a wide temperature range. For example, rainbow trout cells can be grown at temperatures between 5 and 26°C, but even at optimal temperatures (18–22°C), they grow more slowly than mammalian cells at 37°C. A reasonable rate of growth and the availability of incubators are practical considerations that usually dictate the propagation temperature for fish cells. If no incubators are available, most fish cell lines can be grown at room temperature (20–24°C). This will necessitate using basal media that do not require an atmosphere of 5% CO2 for buffering, such as Leibovitz’s L-15. ."

Please re read the bold and italicized sentence. It clearly states that fish cells grown at optimal temperatures grow slower than mammalian cells at 37°C. Nowhere does it state that fish cells can be grown at 37°C. I have worked with zebrafish for last 13 years and have a number of collaborators who use fish cells. I do not know anyone who has successfully grown fish cells at 37°C. The underlined part refers to mammalian cells growing at 37°C, not fish cells. If you still do not understand my point please ask a native speaker of English to explain this sentence to you.

Eminent Scientists, The point made is clear. Let this be a healthy forum. I don't want couple of scientists to have a bitter war of words, I posed this question to have a move on with my research, not to feel bad. Thanks a lot to both Dr. Sandeep Paul and Dr. Rafik Karaman.

Dear Raghunath,

I have cultured goldfish retinal ganglion cell neurons for many years, and have seen zebrafish neurons cultured under conditions similar to ours.  We culture at room temperature in L15, which is an air-buffered media.  Depending on the density of your cells, you may need to ass dome HEPES buffer to the media to maintain the pH.

The lab made its own incubators by buying polystyrene boxes that had loose fitting lids to keep air exchange, and then spray-painted the outside black to bock the light.  Even though we have done this, we still keep the incubator chambers inside a dark room (better for air circulation) or inside of a dark cabinet.

We made tissue culture dish supports to fit the inside of the chambers by cutting polystyrene egg crate to size.  It comes in large sheets since it is used to cover fluorescent light fixtures, and it cuts easily with a hacksaw.  We place the support on top of sterile towels that we have saturated with sterile water in order to maintain the humidity in the chamber.

Since I work on primary neurons, I am not familiar with any cell lines.  However, you may be able to get some information from ZFIN.org, the zebrafish model organism database.

Good Luck!

Jill

Dear Raghunath,

I shouldn't try to type fast late at night.  The first paragraph should read "you may need to add some HEPES"!  A creative change on the part of my spell-check, though.

Cheers!

Jill

Thanks a lot Jill Miotke. You are spot on with your spell check.