endothelial cell isolation from mouse - using magnetic dynabeads from DYNAL (Invitrogen) (Mar/01/2007 )

Hello,

I am trying to isolate endothelial cells from the heart and the lungs of mice, that have compromised levels of the p53 tumor suppresor gene.

I am using magnetic dynabeads for the isolation technique. it is a very crude method of isolation. i extract the organ and then digest it using collagenase II and then try to mince the tissue after digestion using a cannula and then by tituration (using a syringe). I filter the crude extract and the cell suspension I get, I incubate it with the beads which I coat overnight with anti-PECAM-1 Abs. since the endothelial cells express PECAM-1 constitutively, the beads with the anti-bodies, should ideally go and bind to the cells of interest, from the cell suspension. the endothelial cell attached to the magnetic beads are recovered using a magnet.

The problem I am having is I see contamination of endothelial cells with fibroblasts and smooth muscle cells. I tried to use a different cell culture medium, which was high glucose DMEM, but without the amino acid L-valine. I add D-valine instead, since the fibroblasts cannot metabolise this form of the amino acid and hence will not survive.

But I still see fibroblast contamination.

Could someone point where I could be going wrong!!!

pmaj

Have you noted if that contaminating fibroblasts and smooth muscle cells are growing surrounding PECAM+ cells? Perhaps they still attached to endothelial cell during magnetic purifaction if digestion is not enough. Maybe if you try a different digestion method (maybe more time in Col-II) could help to obtain a pure population of endothelial cells.

Good luck!

Hello,

I am trying to isolate endothelial cells from the heart and the lungs of mice, that have compromised levels of the p53 tumor suppresor gene.

I am using magnetic dynabeads for the isolation technique. it is a very crude method of isolation. i extract the organ and then digest it using collagenase II and then try to mince the tissue after digestion using a cannula and then by tituration (using a syringe). I filter the crude extract and the cell suspension I get, I incubate it with the beads which I coat overnight with anti-PECAM-1 Abs. since the endothelial cells express PECAM-1 constitutively, the beads with the anti-bodies, should ideally go and bind to the cells of interest, from the cell suspension. the endothelial cell attached to the magnetic beads are recovered using a magnet.

The problem I am having is I see contamination of endothelial cells with fibroblasts and smooth muscle cells. I tried to use a different cell culture medium, which was high glucose DMEM, but without the amino acid L-valine. I add D-valine instead, since the fibroblasts cannot metabolise this form of the amino acid and hence will not survive.

But I still see fibroblast contamination.

Could someone point where I could be going wrong!!!

pmaj

Have you noted if that contaminating fibroblasts and smooth muscle cells are growing surrounding PECAM+ cells? Perhaps they still attached to endothelial cell during magnetic purifaction if digestion is not enough. Maybe if you try a different digestion method (maybe more time in Col-II) could help to obtain a pure population of endothelial cells.

Good luck!

QUOTE (p.maj @ Mar 1 2007, 07:09 AM)

Hello,

I am trying to isolate endothelial cells from the heart and the lungs of mice, that have compromised levels of the p53 tumor suppresor gene.

I am using magnetic dynabeads for the isolation technique. it is a very crude method of isolation. i extract the organ and then digest it using collagenase II and then try to mince the tissue after digestion using a cannula and then by tituration (using a syringe). I filter the crude extract and the cell suspension I get, I incubate it with the beads which I coat overnight with anti-PECAM-1 Abs. since the endothelial cells express PECAM-1 constitutively, the beads with the anti-bodies, should ideally go and bind to the cells of interest, from the cell suspension. the endothelial cell attached to the magnetic beads are recovered using a magnet.

The problem I am having is I see contamination of endothelial cells with fibroblasts and smooth muscle cells. I tried to use a different cell culture medium, which was high glucose DMEM, but without the amino acid L-valine. I add D-valine instead, since the fibroblasts cannot metabolise this form of the amino acid and hence will not survive.

But I still see fibroblast contamination.

Could someone point where I could be going wrong!!!

pmaj

Hi:

I had fibroblasts contamination to some of my primary culture as well. I am trying to find a media without L-valine. Which one did you use? Could you please give me the catalog number?

Thanks!

Naixus

Hello,

I am trying to isolate endothelial cells from the heart and the lungs of mice, that have compromised levels of the p53 tumor suppresor gene.

I am using magnetic dynabeads for the isolation technique. it is a very crude method of isolation. i extract the organ and then digest it using collagenase II and then try to mince the tissue after digestion using a cannula and then by tituration (using a syringe). I filter the crude extract and the cell suspension I get, I incubate it with the beads which I coat overnight with anti-PECAM-1 Abs. since the endothelial cells express PECAM-1 constitutively, the beads with the anti-bodies, should ideally go and bind to the cells of interest, from the cell suspension. the endothelial cell attached to the magnetic beads are recovered using a magnet.

The problem I am having is I see contamination of endothelial cells with fibroblasts and smooth muscle cells. I tried to use a different cell culture medium, which was high glucose DMEM, but without the amino acid L-valine. I add D-valine instead, since the fibroblasts cannot metabolise this form of the amino acid and hence will not survive.

But I still see fibroblast contamination.

Could someone point where I could be going wrong!!!

pmaj

Hi there,

I get the media custon made- I order the DMEM, high glucose with pyruvate, but I ask Hyclone( the company) to no add L-valine to it. so the medium had no Valine amino acid. Then I order D-valine from SIgma and add that to my media (keeping the same concentration as L-valine). I add 56.06mg per 500 mL of media. thia number I got from the formulation page of the media. You can get it from the company website, where they give you the formulations of the media.

Then I filter the media through a 20 micron filter and add the required Fetal bovine serum, sodium pyruvate, pen/strep, L-glutamine and non-essential amino acids.

D-valine cannot be metabolised by fibroblasts and hence they dont survive in the culture dish.

Hope this helps.

Good Luck

Pooja

Hi there,

this seems interesting.
I will definitely try to see if the 4deg C temperature can shock the fibroblasts and leave the remaining ECs healthy.

Will let you know how it works

Thanks

Pooja