Protocol for endothelial cell FACSort

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ZeroKelvin
ZeroKelvin's picture
Protocol for endothelial cell FACSort

Hi,
I just discovered this site, it's great that people can offer advice over a forum!

I'm trying to use flow cytometry to sort endothelial cells. Does anyone have a generic protocol? Or better yet, we have the APC-CD31 antibody that I'm using, if anyone has any experience with that can you share it with me please?

Thanks in advance!

Guo-qiang Huang
Guo-qiang Huang's picture
CD31 is not endpthelial

CD31 is not endpthelial specific cell marker. I am wondering what the sorting material you are working with? You are working on human endothelial or mouse endothelial? It is hard to help you without knowing your purpose.

ZeroKelvin
ZeroKelvin's picture
Guo-qiang Huang wrote:CD31 is

Guo-qiang Huang wrote:

CD31 is not endpthelial specific cell marker. I am wondering what the sorting material you are working with? You are working on human endothelial or mouse endothelial? It is hard to help you without knowing your purpose.

Thanks, I have primary mouse aortic endothelial cells that are mixed with fibroblasts. I just want to sort out the endothelial cells to get pure culture to do experiments on. I have not been able to find a FAC sorting protocol specifically suited to primary endothelial cells, I've only found ones for other cell types. I'm mostly interested in:
-what to use for blocking?
-any recommended titration concentrations to try for cd31-apc?
-what kind of staining buffer is best to use for endothelial cells?
Do you have any suggestions?

Thank you.

Guo-qiang Huang
Guo-qiang Huang's picture
I am wondering why you choose

I am wondering why you choose CD31 as the marker, can you change it to CD107a, CD105 or some other endothelial cell specific marker? I think the CD31 sorting will not give you cleaning sorting, since it includes some dendritc cells in the sample.

The protocol for sorting should be the same as the protocol of cell staining except you have to control the sterile condition when staining,i.e, to use the filter steriled antibodies;You don't have to stain the cell on single tube basis, you can stain the cells on bulk based on how many cells you estimate to harvest. Off course the quantity of the antibodies used should be proportionally increased according to cell amount and the manufacture's instruction.

You can used steriled routine cell washing buffer ( contains 2% BSA or 5% horse serum) without Azide, add 5mM EDTA is required for endothelial cell sorting.

Try and let me know if it works

Guy Sovak
Guy Sovak's picture
Hi

Hi
Here are two generic protocols for FACS (direct and indirect)
Both are taken from the abcam protocols on line.
http://www.abcam.com/index.html?pageconfig=resource&rid=10419
I hope it will help.

ZeroKelvin
ZeroKelvin's picture
Guo-qiang Huang wrote:I am

Guo-qiang Huang wrote:

I am wondering why you choose CD31 as the marker, can you change it to CD107a, CD105 or some other endothelial cell specific marker? I think the CD31 sorting will not give you cleaning sorting, since it includes some dendritc cells in the sample.

The protocol for sorting should be the same as the protocol of cell staining except you have to control the sterile condition when staining,i.e, to use the filter steriled antibodies;You don't have to stain the cell on single tube basis, you can stain the cells on bulk based on how many cells you estimate to harvest. Off course the quantity of the antibodies used should be proportionally increased according to cell amount and the manufacture's instruction.

You can used steriled routine cell washing buffer ( contains 2% BSA or 5% horse serum) without Azide, add 5mM EDTA is required for endothelial cell sorting.

Try and let me know if it works

Guoqiang, thank you for your suggestions, I agree cd31 is not the most specific but it's what we have in lab. I'll look around to see if anyone has cd105 or cd107a.

And thanks to gsovak, I've looked at the abcam protocols and several others from internet and i just combined/modified them to make my own. Hopefully it works.

Docmagus
Docmagus's picture
You might also want to look

You might also want to look to Tie-2, I have had good luck with cd31, but others have not. BD Pharmagen makes a good antibody for it. Also ddr2 might allow you to eliminate the fibroblasts in a negative sort.

eduardos
eduardos's picture
I had the same problem

I had the same problem (fibroblasts contamination over ECs).
I am willing to try FACSort by using a couple of antibodies... In the end does anyone have a protocol that will not mind to share with me?

Nevertheless, I read that Geneticin is able to eliminate fibroblasts in culture. I will try that also in my primary ECs cultures and I can latter update you guys about it.

Cheers

Yanivr
Yanivr's picture
ZeroKelvin wrote:Hi,

ZeroKelvin wrote:

Hi,
I'm trying to use flow cytometry to sort endothelial cells. Does anyone have a generic protocol? Or better yet, we have the APC-CD31 antibody that I'm using, if anyone has any experience with that can you share it with me please?

Thanks in advance!

Good morning,
A year after ZeroKelvin posted this message, I encounter the same problem - how can I use the flow cytometry in order to sort (bovine) endothelial cells I extract into a pure sample to use in experiments?
I would greatly appreciate any FACS protocols or recommendations on markers.

Thank you.
Yaniv

klsurekha
klsurekha's picture
Guo-qiang Huang wrote:I am

Guo-qiang Huang wrote:

I am wondering why you choose CD31 as the marker, can you change it to CD107a, CD105 or some other endothelial cell specific marker? I think the CD31 sorting will not give you cleaning sorting, since it includes some dendritc cells in the sample.

The protocol for sorting should be the same as the protocol of cell staining except you have to control the sterile condition when staining,i.e, to use the filter steriled antibodies;You don't have to stain the cell on single tube basis, you can stain the cells on bulk based on how many cells you estimate to harvest. Off course the quantity of the antibodies used should be proportionally increased according to cell amount and the manufacture's instruction.

You can used steriled routine cell washing buffer ( contains 2% BSA or 5% horse serum) without Azide, add 5mM EDTA is required for endothelial cell sorting.

Try and let me know if it works

Quote:

Hello, thanks for all this info. I want to purify endothelial cells from mixed population using FACS. I have stained endothelial cells using CD105 ab. Even then I was unable to sort them as there are more number of RBC in my culture. I have lysed RBS afterwards, but even then after centrifugation, I got a red coloured pellet. Basically I am using chick chorioallantoic membrane for isolating endothelial cells and I am mechanically disrupting the membrane to procure the cells. Is there any protocol to get rid of platelets, smooth muscle cells along with RBC and fibroblast cells?
Jason King
Jason King's picture
You need to get rid of the

You need to get rid of the RBCs before doing the FACsorting. I find that I have to use quite a large volume of red cell lysis buffer (ie a 50mL tube rather than a standard FACs tube. 10 minutes incubation on ice , centrifuge and check the colour of the pellet. If it is still red / pink, then resuspend again in RBC lysis buffer. It should work by the second time. Then wash well with PBS and do the FACs antibody incubation(s).

Have you considered negative selection of the macrophages and dendritic cells (probably with the same antibody)? This should leave you with muscle cells, endothelial cells and fibroblasts and I'd expect a CD31-specific antibody to pull out just the endothelials.

Alex S.
Alex S.'s picture
Hey folks,

Hey folks,

Im new to this forum so i just want to say "Hello from Frankfurt/Germany" first.

Im also interested in Endothelial cells and also trying to establish FACS sort of endothelial cells from mouse lung. I decided to use CD31-FITC and CD45-PerCP (both from BD) to distinguish between EC´s and Macrophages but then i found outl, that platelets do also express CD31, which leads ot contamination. But the tip with Tie2 ist very good as we already have a Tie2-PE AB eBiosciences which works perfect in cell culture. But i have a more severe problem. If i dissociate the mouse lung and Sort them i cant find any cells at all in the well. Im using a collagenase D digestion followed by mechanic dissociation of the tissue and i have a good viability after the preparation as determinded by Trypanblue staining. Then i sort them using a BD FACS Aria cell sorter with a 70µm nozzle and 35PSI pressure and it seems they all die. I sort directly in 6wells coated with rat tail collagen and filled with media. I cant even find dead cells just fragements.
So i thought i did something wrong with drop delay etc and i sorted beads to check my handling and everything was alright.
Maybe there is somebody having an idea to that.

regarding Fibroblast contamination. A harsh way to get rid of them from immortalized EC´s is to wash them every day with cold PBS. And to wash means to pull directly the PBS on the cells, again and again. As the EC´s are much more adherent only Fibroblast will be flushed away. After some weeks you should have a pure culture.

So far
Alex

Kate Ploum
Kate Ploum's picture
 Hello Alex,

 Hello Alex,

I am sorry I wont give you an answer to what you are asking..you see, I am just starting as well doing some staining for CD31 for FACS analysis and i am trying to find the optimal digestion conditions. I am working on a different tissue (placenta) but it would be really helpful to me if you told me what digestion enzyme you used, in what concentration and for how long and if your CD-31 staining worked, it meas that this digestion treatment doesnt affect your antigen.

Thank you very much in advance for your help,
Best,
Kate

cellAlchemy
cellAlchemy's picture
 Guo-qiang: What exactly do

 Guo-qiang: What exactly do you mean by EDTA is required for endothelial cell sorting? Can you explain further?