I recall that monocyte isolation has become a topic several times, but I am not sure which is the best way. It maybe depends on the purpose of each experiment. Is anyone out there actually who has compared different methods considering purity, damage (physically or functionally), or stimulation? Here is the methods I have experienced and known of. - Traditionally take adherence cell: 50-60% Monos, of course they are stimulated, but the scatter plot (FSC vs SSC) is better (tighter) than MACS isolated monos. - MACS, http://www.miltenyibiotec.com/index.php Positive selection < 88% (never reached 90%) Negative selection, much lower purity (forgot it because it was not useful for us) Isolated monos sometimes show a spread-out, rough scatter plot on FACS. They are stimulated, and perhaps damaged. They could not survive well in culture (the culture was performed by a grad student, not by me. Please comment if you succeeded). - Hydrophobic culture bag, Cheryl Guyre, JIM 2002 "Advantages of hydrophobic culture bags over flask for the generation of monocyte-derived DC......." This article must be on the very recent JIM. I have the in-press version. According to a co-author, it was better purity, less stimulation, and softer adhesion. - Parcoll: http://memorias.ioc.fiocruz.br/952/3879.html I have not tried yet. It says 90% of purity. Anyone tried? - Sorter: Purity must be the best, but seems it depends on an instrument and setting technique. Does anyone have data regarding the functional assays, recovery rate, % of death on day 0,1,2....? I appreciate any return. Thank you, Mayumi Kataoka FACS Facility Temple University Philadelphia, PA, USA --- Annette Byrne <AByrne@pcyc.com> wrote: > Hi Flowers > > I'm looking for some advice on the best way of separating out the > monocyte > population from peripheral blood and also how does one go about > activating > them to become terminal macrophages ?. > > Also for those of you in the know, is this the best system to look at > human > Macs. To date I've been using PMA activated THP-1 cells but am trying to > get > a more " physiological" way of doing things > > many thanks as always > > Annette > > Annette Byrne PhD > Scientist > Pharmacyclics > 995 E Arques Ave > Sunnyvale > CA 94085-4521 > USA > > Tel: 001-408-3283640 > Fax: 001-408-3283689 > e mail : AByrne@pcyc.com > www.pharmacyclics.com > > > __________________________________________________ Do you Yahoo!? Yahoo! Mail Plus - Powerful. Affordable. Sign up now. http://mailplus.yahoo.com
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