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Message | User | Date(yyyy-mm-dd) |
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medium for 293T Cell line | kavitha | 2008-10-29 | Click here to register. |
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| im using DMEM for the 293T cells. they dont growing fast. can u please tell me what medium could be used for 293T and the components of DMEM. | | |
| lubrol | 2008-10-29 | Click here to register. |
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 | You don't say if you are using serum or at what concentration; typically these cells are grown with 10% v/v FBS. Also, an additional 2mM L-glutamine above what is usually found in high glucose DMEM with L-glutamine and sometime sodium pyruvate will help with growth in many cases. After that, what are your growth conditions: temperature, CO2, plating density, feeding schedule, culture device (plate, dish, flask?) and what do the cells look like and how do they behave? Do they initially adhere but not divide, not adhere, round-up and detach, what color is the medium, have they been checked for mycoplasma contamination? Are the cells come from a high passage number or are they relatively young?
Sorry about all the questions, but more information is better then less in trying to determine where the problem might lay.
You can find the classical formulations of high glucose DMEM with L-glutamine at any of the following links. You can also find out base culture conditions for many cell lines at www.atcc.org (search for the cell line - http://tinyurl.com/5zmsf5 in your case)
http://tinyurl.com/6l437k
http://tinyurl.com/5ohhs2
http://tinyurl.com/6kcfqe
Hope this helps some…
Lubrol |
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| kavitha | 2008-11-04 | Click here to register. |
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| Hi lubrol
thanks for your valuable suggestions.
This is my culture work conditions im using to grow 293T cell line,
10% FBS,
2mM glutamine,
temperature 37'C,
5% Co2,
T25 flask,
subculture - 1:2 ratio,
I could see clumps of cells rather than separate cells,
they adhere,
the medium color is pinkish orange,
i could not find significant morphology,
can u please suggest me whether i should increase the sodium pyruvate concentration in the medium (now i'm using 110mg sodium pyruvate/liter ) |
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| lubrol | 2008-11-06 | Click here to register. |
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| Kavitha;
I don't think that you need to increase the pyruvate concentration unless the medium is fairly old - more than a few days after having the serum added. You might want to add another 2mM L-Glutamine as it is can be helpful. But I'd try the trypsinization tips below first.
Based on the color you report the pH is about correct.
Because you report clumping I'm wondering how you are dissociating the monolayer to subculture it: with a 1:2 ratio either you are seeding at a fairly high density or subculturing sub-confluent flasks. I'd make sure that I rinse (I prefer Hank's to Dulbecco's, but it doesn't make a lot of difference the flask with calcium and magnesium free buffer and possibly with 0.2 g/L EDTA in it as well. Keep the trypsin exposure as short as possible and keep everything at 37° C. [These cells really don't attach well if they have spent any time at room temperature (25° C) - this is a real common issue after thawing from liquid nitrogen and can take several days for the cells to adhere well.]
After rinsing the cell sheet with the buffer solution to make sure all of the serum proteins are gone, you can try incubating for about 2-5 minute with the EDTA containing solution before adding the trypsin solution. The trypsin solution shouldn't be left on for more then 2-3 minutes. If the cells don't appear to be coming off well, increase the time they are exposed to the EDTA containing buffer solution instead of increasing the length of time you use the trypsin. As the cells begin to detach you can gently rock the flask from side to side (about 15-20 seconds) to encourage the cells to come off and then pull the suspension very slowly up and down a couple of times in a sterile pipet. Remember to neutralize the trypsin by adding about 5-8 ml of complete medium (has to contain serum) to the flask. If you spin the suspension down, keep the g-force low (no more than 1,000 RCF) and for only a couple of minutes: it doesn't take as long as many people think and even if you loose some cells, the cells in the pellet will be happier for seeing less g-force for a shorter time. It is not always necessary to spin down the cells, if you keep the volume of Trypsin solution to around 3 ml in a 75 cm² surface area flask you can split directly into new flasks - really just enough to cover the monolayer completely.
Hope this helps; unfortunately, these are not the easiest cells to use; the viral transfection has caused some issues with growth.
Lubrol |
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