Have you supplemented the media? And also as you said they are new so that's why they are taking time to adapt to the media. Continue with the same media.

dear Zara

maybe, your serum is not adapted to these cells, try another one.

Hi Zara,

Usually newly cultured cells need to be passaged a number of times until they are able to fully adhere to your plate/flasks and adapt to their new environment. In order for cells to grow and become confluent with RPMI 1640 many researchers would add a growth factor such as Fetal Bovine Serum (FBS - 5% to 10%) or Insulin Transferrin Selenium (ITS 0.6% to 0.8%). Another aspect that needs to be considered is the tempreture that you have set your incubator to. Most of the cells that I have used grow at 37oC (Human body temperature) however this may be different for your cell lines.

Attached is an article, which provides an example of a cell culturing protocol that was used in order to reach confluency for your cell line.

Article Effect of sirolimus on urinary bladder cancer T24 cell line

Also, I have provided a website which illustrates the seeding densities required for each type of plate and flask in order to reach the confluency you require

https://www.thermofisher.com/au/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html

With regards to the colour of the culture medium RPMI 1640 contains phenol red which is a pH indicator. When the medium is becoming acidic it will begin turning an orange-yellow colour. This is because the cells are consuming all the substrates from the media and producing acidic by products. I would recommend changing the media when it turns this colour.

Hope this helps

Dear Zara,

I worked with these cell lines for several years. These two cells grow very well, Initially it is slow, but later it grow very well if not contaminated with mycoplasma. So far I remember, I used RPMI 1640 for 5736 and DMEM for T24 cells. 10% serum is fine and you don't need anything else. 5736 cells is very sticky to culture plate, very careful for passaging!!! If you need any further help, please mail me: [email protected]