Splitting A549 cells - (Jul/27/2011 )

I am new to cell cultures, and I have been assigned the task of splitting someone's A549 cells..
I have a T75 flask that I was told to split into 5 flasks, and add 30 mL of media to each.
I just wanted to verify something-if I split 1:5, I re-suspend the cell pellet in 5 mL of media and add 1 ml of the cell/media solution to each of the flasks? I am trying to properly grasp the concept of splitting cells in certain ratios, so if you someone could give me some examples that would be great and much appreciated!

Thank you

biochem88 on Wed Jul 27 20:23:45 2011 said:

hey biochem88...welcome to Bioforum. yup, that's about right...and I guess they're adherent cells so you probably have to trypsinise them first?

casandra on Wed Jul 27 20:36:09 2011 said:

Yes!

biochem88 on Wed Jul 27 20:36:48 2011 said:

Yes to what? And you've got very good sterile technique? esp no coughing, sneezing, and scratching onto your cells....that's more important than doing simple division ...

casandra on Wed Jul 27 20:42:00 2011 said:

Yes to they are adherent cells so we trypsonize them first. I've watched people work for the past two weeks and have worked with the cells a few times while other people watched over me, plus I've watched a lot of basic cell culture technique videos! I've just been trying to learn more about the fundamentals of cell growth, splitting etc because even though I have been observing people work, I don't necessarily understand "why" certain things are done - most of my research so far has been in protein purification and analysis, and not cell cultures.

biochem88 on Wed Jul 27 22:23:23 2011 said:

I think your strategy is sound and you learn a lot by watching how people work. How you split cells would depend on many factors eg is it just for maintenance of the cell culture, for an experiment in one or two days or to skip coming to the lab on the weekend? It depends also on your cells...for slow growing ones, better to use lower split ratio as opposed to fast growing ones (to avoid overgrowth)...so know your cells..check all the relevant info..and cultivate/perfect your aseptic technique....and when in doubt, always consult more experienced people from your lab....or from here...like Uncle Rhombus or Bob1(not Sponge)....

Just an observation, 30ml seems like a heck of a lot of media for a T75 flask for adherrent cells.

leelee on Thu Jul 28 04:41:16 2011 said:

I think I have read that the volume range for a T75cm2 flask is 15-37.5ml for optimal CO2 penetration.
Also, they are going to be growing over the weekend.
It was just what I was also told to do by the person growing the cells.

What I do with A-549 Is the following.

A T-75 95% - 100% (almost 3.52e5 cells in 10ml) could be divided by 3 T-75 with 20 ml of media and 2 days later you have 3 confluent T75 flasks, and could be divided on 9 flasks.

I hope this would be helpful for you.

biochem88 on Thu Jul 28 13:23:56 2011 said:

Dear Biochem88,

You have read it in Ian Freshney's Culture of animal cells which is the "Bible" for anyone who wants to do/or is doing cell culture.

He states that the volume of culture media should be within the range of 0.2ml-0.5ml/square centimetre i.e. for a T75 flask 15ml-37.5ml .......this is dependent upon the confluency of the cells.

Split ratio's are just a guide and are sometimes flexible. However you should never exceed the maximum split ratio as the cells may not ever grow to confluence

Hope this is useful

Kindest regards

Uncle Rhombus