Hi Hadi, When you titrated your antibody using only a million nucleated BM cells and found out that 2 ul of antibody in 0.1 ml solution works best, you determined the proper antibody concentration. The total amount of antibody molecules in 2 ul is generally several fold more than the number of available antigens and can therefore also be used to label 10^8 nucleated BM cells ... as long as you keep the antibody concentration the same and therefore the total volume the same. In your case, it means you need to make a cell suspension in 98 ul (~10^9 cells/ml) and add 2 ul of antibody. For confirmation sake, you should ask the antibody manufacturer what the antibody concentration in the vial is (1 ug/ml ?) and you can do the math to determine how many antibody molecules you're using. I do this all the time and it works great. In fact, using very high cell concentrations (and minimum amount of antibody molecules) give much better results because it reduces background labeling. By the way, this topic has been discussed several times on this list before. A quick search will give you additional info. Good luck, Ruud -- <http://www.cytonome.com> Cytonome <http://www.cytonome.com> Dr. Ruud Hulspas, Ph.D. Director of Cytometry Cytonome <http://www.cytonome.com> 27 Drydock Avenue Boston, MA 02210 phone: 617-330-5030 x226 fax: 617-330-5031 Ruud-Hulspas@cytonome.com <mailto:ruud-hulspas@cytonome.com>Received on Thu Jun 26 20:38:00 2008
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