Hi,

I use the ImageStream (amnis corp.), an imaging flow cytometer. It can read up to 5000 cells/sec and and can capture high-resolution images of up to 10 fluorescent channels as well as bright field images. You can define your population by gating, as in conventional flow cytometry, and get high resolution images of these cells. You can see an example in one of our projects:

http://www.nature.com/ni/journal/v13/n11/full/ni.2408.html

Good luck,

Ziv

Article Monocytes-macrophages that express ??-smooth muscle actin pr...

the most difficult and the easiest way at the same time-staining of live cells :)

It is also possible the application of the method of light microscopy ( bright field method)

tissue immunofluorescence would be the way if you cannot do flow but this would require: 

(1) you have a working antibody for the rare cell population

(2) test if the epitope for your antibody works in cryosections or paraffin sections

(3) if it is rare, you might have to look through hundreds of sections before you can capture an image and this would be really hard to quantify

You might also want to consider live staining fluorescent dyes that is fixable (ROS, DNA content, mitochondrial dyes etc). Rare cell = not cycling? (so BrdU pulse or cell cycle dyes/antibody based staining).

Hi Fatemeh, are you looking at circulating tumor cells (CTCs)?

If your detection system is based on antibodies, you could also try using magnetic sorting (MACS) to either deplete negatives or harvest positives. That way you would concentrate your population of interest

thanks a lot...

Dear Steingrimur Stefansson

yes i am working on CTCs

Hi Fatemeh, the FDA approved method of analyzing CTCs is by using the CellSearch system. However, this technology only allows for CTC enumeration. This assay uses mag. particles coated with anti-EpCAM antibodies to capture CTCs and identify them by DAPI and anti-cytokeratin 8, 18 and 19 Abs. Anti-CD45 Ab is used to exclude leukocytes.

There are commercially available filtration methods that isolate CTCs based on size and allow you to stain them with Abs of your choice. .

 http://www.screencell.com/

http://www.creatvmicrotech.com/ctc.html

Hi,

I use the ImageStream (amnis corp.), an imaging flow cytometer. It can read up to 5000 cells/sec and and can capture high-resolution images of up to 10 fluorescent channels as well as bright field images. You can define your population by gating, as in conventional flow cytometry, and get high resolution images of these cells. You can see an example in one of our projects:

http://www.nature.com/ni/journal/v13/n11/full/ni.2408.html

Good luck,

Ziv

Article Monocytes-macrophages that express ??-smooth muscle actin pr...

see our paper for one approach

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0078261

We had this question for a while over 20 years ago for a population almost as rare as CTCs. I am speaking about CECs, 1 to max 10 CEC/mL blood. We succesfully "played" with a very specific MAb (S-Endo1 aka CD146) and immuno-magnetic beads (Dynabeads). You can use Acridine Orange to see the brighly fluorescent isolated ("rosetted") nucleated cells. This is still in use nowadays although fluorescent microscopy seems (and does remain) a tedious task. in this paper we used intra-cytoplasmic staining of VWF to help characterizing endothelial cells but later the fluorescent lectin (Ulex) which binds the surface, has also been used for counter-staining the "rosetted" isolated cells.  

See: Thromb Haemost. 1992 Jan 23;67(1):147-53.

Rapid isolation of human endothelial cells from whole blood using S-Endo1 monoclonal antibody coupled to immuno-magnetic beads: demonstration of endothelial injury after angioplasty. George F, Brisson C, Poncelet P, Laurent JC, Massot O, Arnoux D, Ambrosi P, Klein-Soyer C, Cazenave JP, Sampol J.

If you want more litterature on that, search for CEC (circulating endothelial cells), S-Endo1, CD146, IMS as keywords and/or George F (or Dignat-George F, present on RG) and/or Woywodt (among others).

I fully agree with Steingrimur Stefansson that the CellSearch system has been developped and approved for CTC detection but this is an IVD system possibly too expensive for the research use you seem to present in your question.

The S-Endo1 MAb is available from BioCytex for coupling on M450 anti-mouse Ig Dynabeads.

In case FCM would be your preferred bet, you may want to read a more recent approach using pre-enrichment with S-Endo1-coated Ferro-Fluids (the same as in Cell-Search) before differential FCM analysis.

See: J Thromb Haemost. 2008 May;6(5):869-76.

CD146-based immunomagnetic enrichment followed by multiparameter flow cytometry: a new approach to counting circulating endothelial cells.

Widemann A, Sabatier F, Arnaud L, Bonello L, Al-Massarani G, Paganelli F, Poncelet P, Dignat-George F.

You may want to try pre-enriching CTCs using the magnetic beads of Cell-Search and adapt your FCM protocol using CD45 (a leukocyte exclusion marker), an appropriate cell-permeant nuclear dye (e.g. Syto ??... Molec. Probes) plus whatever cancer-associated marker(s) that may be highly expressed on your target rare cells (e.g. Muc-1, EpCAM again, EGFR, HER-2 ...). Good luck !

Alternatively, if you do not want to mount your own method but you are looking for finished products/R.U.O. kits, you can find also tools/ kits in the Miltenyi portfolio, as also suggested above.

Hi Philippe, this is off topic for this thread, but have you seen autofluorescent vesicles in CEC's or other Endo's?

https://www.researchgate.net/post/Autofluorescence_vesicles_in_HUVEC_cell_Lines

If you have very rare cells you can try to spin them onto slides and view them under the microscope. I have recently tried DeNovo's Jetta100 microfluidic device and found that it enriched my rare cells (they are bigger than blood cells) and so I could stain them and view each individually under the microscope.

good luck!