Scientist Solutions: International Life Science Community By Scientists For Scientists
    
Home » Forums » Cell Culture and Tissue Culture » Transfection and Assay Development » HEK293 cells detach after transfection

Thanks to our sponsors who make this site possible

HEK293 cells detach after transfection

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.

HY
United States

Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Topic Started by HY
on 10/20/2009 13:38 PM   
Reply to this post Go to the top of the page

Hi,

I start working on HEK293 cells recently. I transiently transfect HEK cells with plasmids which encode an ion channel. I found many cells detach easily after 6h incubation with lipofectamine 2000.  My cells are 60-70% confluence before transfection. I use 1.5 ug plasmids in 200 ul MEM, and 5 ul lipofectamine 2000 in 200ul MEM. MEM has no antibiotics or serum,   I record whole cell current one day after transfection. My data are not good at all.

So, I am wondering whether cell lose after transfection affect data intergrity. Is there any good way to manage HEK cells, letting them adhere tightly to the dishes?

I would appreciate greatly for your valuable suggestions.

HY


Replies
Pippuri
United States

Send PM
See Mini bio

Status: Protein Chemistry & Glycobiology Moderator
Frog Laureate
Posted By Pippuri
on 10/20/2009 14:28 PM   
Reply to this post Go to the top of the page

 Hi HY,

One thing about using lipofectamine 2000 is that, the culture needs to be >85% confluence, or else it tends to kill cells quickly. Thus, I suggest you to start with a bit higher cell density. HEK293 is really easy to transfect once you catch the right condition. As I am not sure the size of your culture dish, please double check the amount of DNA per size on the instruction sheet to make sure that your DNA amount is within the range of the company's suggestion. Having too much DNA will also kill the cells (I learnt it the hard way).

I do notice that HEK293 cells won't attach tightly to the culture dish comparing to other cell types... The only trick that I have is to avoid adding media directly on top of the cells. Instead, gently add the liquid from the side wall. This should help to minimize cell loss...

Good luck!


Last edited Oct 20, 2009, 16:51 PM by Pippuri

HY
United States

Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Posted By HY
on 10/20/2009 22:48 PM   
Reply to this post Go to the top of the page

Hi, Pippuri,

Thank you very much for your information. I use 6-well plate for the experiment. I did add media from the side wall. But my cell confluence is defientely lower than 85%.I will try to increase cell confluence next time. Thanks again.

HY



ebola
Australia

Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Posted By ebola
on 11/14/2009 6:48 AM   
Reply to this post Go to the top of the page

 Hi

I started to work on HEK293 derived cells line with some inducible gene expression few weeks ago. We got the cell lines from oversea and we had similar problem of cell detachment during infection with virus and the fixation step. We had minimised the cell detachment problem during infection step by seeding HEK293 cells on collagen-coated plate beforehand (got this idea from some other people's experience on working with stem cells). I guess this might be helpful to your situation. 

Best luck




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.