RE Adipose tissue in flow cytometry

Philippe BOURIN philippe.bourin at efs.sante.fr
Thu Jun 7 04:58:31 EDT 2007


Hello Ann,

The preparation depend upon the type of harvest you have. If it is
dermo-lipectomy, you need to minced the sample in very small pieces, if you
obtain adipose tissue from liposuction you do not need to further cut the
tissue. Wash 2 to 5 times the pieces of tissue to eliminate the red blood
cells. Then you have to digest the preparation. The favored digestion
method is collagenase (Liberase Hi from Roche work very well, but it is
quit expansive). After you must centrifuge the digested samples. The upper
fraction contains the mature adipocytes, the pellet contains the stromal
vascular fraction (which contains the hematopoietic cells). Then you must
filtrate the SVF with cell strainer (BD) 100µm and after 40 µm. At this
step, you obtain a cell suspension without debris containing around 50% of
CD45+ cells. I think you do not need a density gradient separation before
sorting. If you want you could perform a RBC lysis.

Good luck,

Philippe BOURIN
EFS-PM
Laboratoire de thérapie cellulaire
75 rue de Lisieux, 31300 Toulouse
tel: 05 34 50 24 78
port: 06 84 58 08 21
fax: 05 34 50 24 70


								       	     "Ann Kelly"					       	     <kellya at ohsu.edu>					       								         A 
	     06/06/2007 19:16	       Cytometry Mailing List	       				       <cytometry at flowcyt.cyto.purdue.edu> 
								        cc 
								       								     Objet 
				       Adipose tissue in flow cytometry								       								       								       								       								       								       


Hello Flow-ers,
   My principal investigator is looking for information from those
researchers with experience in the analysis of adipose tissue via flow
cytometry. This is a new area of research for our group, and we are seeking
a method for cell separation. Our hope is to be able to isolate lymphocytes
and granulocytes from the whole of the adipose tissue.  Here are a couple
of basic questions:
 1. What method of tissue digestion is favored prior to a cell sort?
 2. Do we need to sort the granulocytes and lymphocytes from the other
cells present in adipose tissue with a density gradient ( i.e. sucrose,
Percoll, etc.) prior to performing a cell sort?

Any advice about our new endeavor is welcome.

Thank you,
Ann Kelly
Oregon Health Science University
Senior Research Assistant
Department of Pulmonary and Critical Care Medicine
-------------- next part --------------
HTML attachment scrubbed and removed
-------------- next part --------------
A non-text attachment was scrubbed...
Name: graycol.gif
Type: image/gif
Size: 105 bytes
Desc: not available
Url : https://lists.purdue.edu/pipermail/cytometry/attachments/20070607/5b34923d/graycol-0002.gif
-------------- next part --------------
A non-text attachment was scrubbed...
Name: pic63968.gif
Type: image/gif
Size: 1255 bytes
Desc: not available
Url : https://lists.purdue.edu/pipermail/cytometry/attachments/20070607/5b34923d/pic63968-0002.gif
-------------- next part --------------
A non-text attachment was scrubbed...
Name: ecblank.gif
Type: image/gif
Size: 45 bytes
Desc: not available
Url : https://lists.purdue.edu/pipermail/cytometry/attachments/20070607/5b34923d/ecblank-0002.gif


More information about the Cytometry mailing list