Immortalized human podocytes - (Dec/06/2011 )
Does anyone have any experience of working with immortalized human podocytes?
I am currently working with the AB8/13 podocyte cell line.
Happy New Year and thanks for your reply. I have so many questions!
I expect you're very busy but could I ask whether this cell line was purchased or did you get it directly from Moin Saleem's lab?
I have inherited some immortalized human podocytes which I have been working on. I'm not sure whether they are in great shape and have a horrible feeling that they may be contaminated with mycoplasma. I have read various things which suggest that podocytes can be a bit problematic...my cells appear to differentiate by around day 14 at 37 degrees but I suffer huge cell loss. I was using a nephrin Ab from Sigma which should detect nephrin at 100kDa (WB) but I consistently had a signal at 75kDa, I did write to Sigma but didn't get a response. I have since heard that there isn't a good nephrin anti-body commercially available. I've heard that Sigma do a good podocin anti body and it appears that Santa Cruz also seem to be popular for anti bodies to podocyte markers such as WT1, CD2AP and synaptopodin. Do you have any preferences?
I would be so grateful for any advice or pearls of wisdom! Unfortunately, I don't have access to anyone with enough specialist knowledge so I'm really struggling.
Hi Pods! Happy New year to you too.
We got the cell line from Moin Saleem 's lab directly. We have them differentiated for 14 days at 37 C just like you and refresh their medium frequently. Which medium do you use? We use RPMI 1640 supplemented with 10% FBS, 1% penstrep and 1% ITS.
I will ask a colleague who is currently working with nephrin and give you more info on that. Anyway, there are some papers out there demonstrating that AB8/13 fail to express podocyte specific markers at times and I advice you to spot them in case your experiment depents on such proteins.
This is Simi and am doing my PhD in India. I am working on mouse podocytes and I would like to ask you the protocol for transfection in podocytes. I tried lipofectamine method, but the cells do not look healthy and I tried microporation but very few cells get attached. Please send me the protocol of how you do transfection in podocytes.
It will be great help for my project.
Thanks a lot