Scientist Solutions: International Life Science Community By Scientists For Scientists
Home » Forums » Cell Culture and Tissue Culture » General Tissue Culture » split ratio, PBS wash, trypsination problem

Thanks to our sponsors who make this site possible

split ratio, PBS wash, trypsination problem

RSS Feed

Would you like to save this topic, event, protocol or job so you can find it again easily?

Just click the "Save to My Lab Drawer" link and the item will be saved in the My Lab Drawer section of your bench space.

Available to members only. Please log in or register for your free account now.

fatemeh safari

Send PM
See Mini bio

Status: Tadpole
Topic Started by fatemeh safari
on 4/10/2010 1:18 AM   
Reply to this post Go to the top of the page


1) how can I get the best split ratio for my cells?

2) is it enough to wash the cells with PBS onec, or it's better to do it twice?

3) last time when I tried to do cell detaching, cells were agglomerated to each other, is that because the activity of Trypsin is not enough, or it has an other reason?



Send PM
See Mini bio

Status: Tadpole
Posted By tanyag
on 4/12/2010 10:56 AM   
Reply to this post Go to the top of the page

Hi Fatemeh

1) The best split ratio for your cells really depends a lot on the cells you have. If there is any literature available elsewhere on the cell line you are using, you should probably start with that and go from there. Certainly anything you have from the place you sourced your cells from would be useful. In my experience, what works in one lab, does not necessarily work the same in another even though they would seem to be working in the same conditions.
I would start with a range of cells splits (eg. 1:5, 1:8, 1:10) and see how they like it. If they are confluent in 2 or 3 days, try a larger number (1:20). We aim to split our cells once a week, so we aim for a split ration that will accomodate that, will also keeping our cells alive and happy. Having said that, there are those that prefer to be split more often or require a certain cell ratio. That's where having specific information on our cell line would come in handy.

2) I was always taught to wash the cells twice with PBS. I find that your first rinse will be coloured by the media you are using, while the second rinse will be clear. I suppose that way you are ensuring you have got everything off.

3) Cells will often clump together after trypsinizing. They need to be broken up by aspirating carefully with a pipette or similar item once they have been resuspended.. This will normally not damage the cells. Some need more effort than others.

Hope that helps.

fatemeh safari

Send PM
See Mini bio

Status: Tadpole
Posted By fatemeh safari
on 4/11/2010 22:28 PM   
Reply to this post Go to the top of the page

Hi Tanyag,
Thank you for clear and helpful answer.

As a Scientist Solutions member, you are able to register a positive vote for any topic which you believe is useful and relevant to our board or any reply which you believe is especially well worded and helpful.

By participating in the voting, you will be helping to identify the best topics & replies on the board.

You may vote once for any one post, and you may not vote for your own posts.

A post (topic or reply) will earn one "thumbs up" icon for every 10 votes received (up to 3 thumbs up), and the person who made the post will also earn two bonus points.

learn more about member points.

Click here to
Become a member & join our
community (It's easy & free)
Already a member? Please log in
User Name  
Forget Password?
Not finding the answer you need?

Post a new topic

You must be logged in to post. Log in above.
Not a member yet? Click here to register
(it's free)
Thank You to Our Sponsor