on 11.08.2000 10:21 Uhr, Carolyn Jefferiss at prscmj@bath.ac.uk wrote: Dear Carolyn, I can imagine that you have to digest the information on the fluorescence labeling of MACS separated CD34 cells. > 1) Thanks you so very much, everyone who had replied to my > minimacs/sorting queries, which I have yet to digest properly The answers seemed to be contradictory so I will try to shed some light onto the mystery. >> >> Appenrently CD34 >> labelling after MACS selection does not diminish the fluoresence >> intensity of FITC, PE or APC labelled CD34+ cells. >>> 2. If you are checking the purity of your cells with an antibody recognizing >>> the same epitope as the MACS beads, you will have problems as many or most >>> of the epitopes will already be occupied with the antibodies attached to >>> the beads. You need another antibody which does not compete with the first >>> one. Both considerations were right - depending on the antigen. The MACS MicroBeads work under extremely strong magnetic fields, thus you really need only a few MicroBeads per cell to get a cell retained in a separation column. For e.g. CD4 on human T cells is present at several thousand molecules per cell, only a small proportion of these molecules are occupied by MicroBeads. In such a case it is possible to use the same clone as a fluorochrome conjugate to stain the cells for flow cytometry. In the case of CD34, the antigen is expressed at a lower level and thus the Beads cover already a significant amount of the available molecules. The antibody used for detection should recognize a CD34 epitope different from the epitope recognized by the antibody which is conjugated to our MicroBeads. The antibody clone QBEND/10 used for our CD34 Progenitor Cell Isolation Kit and CD34 MultiSort Kit binds to epitope II of the CD34 antigen. For the staining of MACS isolated CD34+ cells we recommend using an antibody recognizing CD34 epitope III, e.g. clone HPCA-2 (Beckton Dickinson, San Jose, CA) or MACS fluorochrome conjugated antibodies CD34-PE or CD34-FITC (clone AC136). Special recommendations are indicated in the data sheet when necessary. I hope this helps Hans-Peter -- Dr. Hans-Peter Steffens Technical Support Miltenyi Biotec GmbH Friedrich-Ebert-Str. 68 51429 Bergisch Gladbach Tel: +49-2204-8306-264 Fax: +49-2204-85197 http://www.MiltenyiBiotec.com/
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