Monoclonal screening of stable cells - How to maintain it to confluent stage? (Sep/26/2007 )

Hi there,

I'm working on monoclonal screening and had finally seeded a clone into each well in a 96 well plate. Fresh selective media had been added. Now the question...

1. How regular do I need to change the media?

2. Procedure to change the media: Discard half of old media and replace with fresh media or completely discard old media then replace with fresh?

3. How long will a single colony become a confluent state? I hope not 3 to 6 months!!! I'm working on both HeLa and SH-SY5Y.

4. Do I add more FBS to push the growing process, say 10% to 20% FBS? Will cell grow quicker that way?

5. What am I missing? Hope to get some pointers.


Many many thanks.

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every 2 days

i remplace all the medium. But if you think your cells gonna detach, work in 300µl of volume and try to replace 2/3

well in 96 well, as soon as the medium turns yellow, you can put your cell clone iun 6 or 24well plate. It depends of the capacity of your cells to support divison I go directly for 6well plate in general, beacuse it's faster, and if you are in 24well, you'll still need to go at 6well-plate step.

10% is fine

well when transfering from 96well plate pipett 100µl of pure trypsin and pipett up and down several times to help cell detachment and cell separation.

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Hi Fred,

Many many thanks.

Just one last crucial question...

I need to continue changing media for every two-days until.... approximately how long?

I'm asking this because now that I've started the screening process and I'm literally STUCK to the lab for every 2 days, inclusive of weekend, for ???. Sigh... Haven't been home for quite sometime and am wondering now when will I be able to escape all this. Please don't tell me I'll be stuck for more than 2 months, I'll go crazy.

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you change the medium every 2days.
So it will get the moment (1 to 2weeks for Hela cells) when your well will be yellow. Then standard trypsinisation procedure.
Whole process (from clone seeding to final 15cm plate may took a month

ok if you can't come one week end your cells gonna survive (also certain cells which are really tough may not...). If in general your cells can afford 3days without medium change, then you can have a week end

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Many many thanks.


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