Peter, I have had a similar situation. For a monocyte adhesion study, I was trying to load human U937 or THP-1 monocytes, with 7-amino-4-chloromethylcoumarin (CMAC)from Molecular Probes, and the cells all stuck to the walls of the staining vessel. This stain also is incompatible with serum in the media. Serum-less media may make the cells more likely to stick. I was able to solve this problem by making sure the cells were shaking or rotating during the 30 min loading period. Hope this helps. Cissy Geigerman University of North Carolina at Greensboro On Fri, 31 Aug 2001 13:49:07 +0100 Peter Kierulf <peter.kierulf@ioks.uio.no> wrote: > > > Dear Flowers > > Always some problems (probably for many of you). > > For some time we have tried to label human monocytes (cryopreserved and > thawed) with Molecular Probes Vybrant Cell-Labeling Solution. Our major > problem is aggregation of the cells. > > We have followed the protocol carefully, using cells as recomended of 1 x > 106 /mL, without serum, and adding 5 mL of the Cell-Labeling Solution per > mL of cell suspension, incubating for varying times (30 minutes 37oC, > spinning the cells and washing a couple of times. > > Generally the cells have been aggregating or adhering to our plastic tubes > (Falcon Polypropylene tubes 352070). > > Our intention has been to study cell migration in TransWell systems using > this labeling technique. > > All suggestions to circumvent aggregation is most welcome. > > Thanks! > > > Peter Kierulf > > Peter Kierulf,MD PhD. Professor I > Clinical Chemistry Dep. > Ulleval University Hospital > N-0407 OSLO, NORWAY > > Telephone: +47 22 11 94 86 > Telefax: +47 22 11 81 89 > _-----------------------------------Mobil telphone: 922.85111 > E-mail (RFC Formate): peter.kierulf@ioks.uio.no > E-mai. (SA Formate): /G=peter/S=kierulf/OU=ioks/O=uio/PRMD=uninett/C=no ---------------------- Cissy Geigerman Dept.of Nutrition and Food Service Systems Univ. of North Carolina at Greensboro (336)334-5313
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