Date: Thu Jun 07 2007 - 04:58:31 EDT
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@ohsu.edu> A 06/06/2007 19:16 Cytometry Mailing List <cytometry@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
