Isolamento di cellule mononucleate dal Sistema Nervoso Centrale di ratti con EAE

hi

this is liela from iran i am working on my propozal in pastuor institute in tehran .

i and my profesor D.r kadivar isolated mononuclear cell from umbilical cord blood term in hospital end then inject to a 120gr rat for homing,and determine or detect them whit PCR technics but we have some difficulty?can u help me?

 

Hi Liela,

I am not sure I can help but will certainly try. What kinds of problems did you meet with your protocol? How did you inject the cells (intravenously, intraperitonealy)? How many cells did you inject? How long after injection did you look for the cells? Where did you look for the cells?

Many cells will stay stuck in the very thin capillaries of the lungs so you would have to inject several million (5-10 million) to detect them afterwards.

A main concern is the change in species. Did you do anything to prevent the rat's immune system from kiling the human cells you injected? This killing can happen very fast and may be the main reason why you don't detect any cells.

Christine

Hi Christine,

This is an excellent video and your technique is very impressive.

My name is Yoyo, a post-doc at Imperial College, London, UK.

I have been trying to isolate "sub-cells" (probably MNCs) from Rat left ventricles. The PhD student who left now used to just isolating the LV, followed by mincing with razor blades and then digested in buffer containing collagenase II at a shaking water bath (37C). After 4x15min digestion, cells were filtered through both 70 & 40um cell strainers and spun down. Cell pellets were then lysed with RLT buffer containing B-ME for RNA extraction. However, the quality of cells was usually low (determined by Trypan Blue) and thus the RNA yield was rather inconsistent.

When I took over this project, I thought I could use Ficoll gradient to remove dead cells. It seemed to work, but the amount of cells and the quality of RNA were rather low (260/280 ratio was 1.4-1.6). I did more research and found that Percoll would be more suitable if isolating cells of species other than human. I then followed a protocol obtained from another post-doc working at a different campus of Imperial College. Her protocol is as follows: re-suspend cell pellets in 12ml 1.082g/ml Percoll, and prepare Percoll gradient in a 50ml tube loading from the bottom of a total of 12ml each of 1.050 and 1.082 (with cells) g/ml Percoll. The tube is then centrifugated at 2000g at room temperature for 30 minutes. This should result with debris/lipids at the top, the fibroblasts and cardiomyocytes at the interface between 1.050 and 1.082, while the pellets will be red blood cells and more debris. As I still strain my digested cells with both 70 % 40um cell strainers and my mincing with blades should have damaged cardiomyocytes to a large extent, I don't think there should be any or many cardiomyocytes left in the interface.

I would very much appreciate your expertise and help on this isolation. Is it correct to follow her protocol? I think the Percoll gradient of her and yours are similar to each other, except that you overlay cell suspension in 20ml 30% Percoll onto 10ml 70% Percoll while her was not really overlaying and it seems to be 1.050g/ml Percoll to be at the bottom instead.

I would be most grateful if you could give me some advice on this isolation.

I look forward to hearing from you soon.

Cheers,

 

Yoyo

Dear Dr Beeton, first, thanks for your excellent video.Could i ask you to tell me how can i make 30% and 70% percoll and the percentage of PBS.

regards

I am not sure what your question is. These are simple solutions:
For the RPMI + 30% Percoll, you need 30% Percoll and 70% RPMI.
For the PBS + 70% Percoll, you need 70% Percoll and 30% PBS.

Dear Dr Beeton, thanks for your reply.

Hello Dear Christine Beeton
Thanks for your useful video. I would be grateful if you response my question: In this video when you work with Mycobacterium tuberculosis H37RA for making it thin powder, you didn't wear mask, is it safe for you?

Regards

Hello Dear Christine Beeton
I do apologize, above question is about your another video (Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis), but I wrote it in this page, wrongly. I would be grateful if you response me.

Best Regards

Hi Dear Dr Beeton

Could we use Ficoll gradient instead of Percoll for isolation mononuclear cells from the CNS?

I don't see any reason why you couldn't use Ficoll instead of Percoll as long as the gradient densities are maintained.
Christine

I am grateful for your response.

Hello Dear Dr Beeton

I don't access to the your very useful article, so I don't know the speed of centrifuge for two following steps: 1. preparing the pellet of cells in 50ml PBS
2. make the mononuclear cells layer by using percoll gradient

I would be grateful if you help me, it is very necessary for my thesis.

With Best Regards