Dear Sandra and Cheryl, We got experience with this antibody conjugated to PE. The FITC should do in this case. The problem is the very low levels of IL12p70. Since this Ab shows you the homodimer (p40/p40) and the heterodimer (p35/p40) you will see considerable amounts of protein in monocytes stimulated by LPS for instance. Keep in mind that the homodimer is not the biologically active form (but might have a receptor-blocking function). We stain with succes monocytes and dendritic cells with this antibody. And we use 1 µl Ab per 10e5 cells which were fixed in 4% formaldehyde (in PBS) and permeabilised with 0.3% saponin. To stain the biological effective protein (p70) use the clone 20C2 (Pharmingen 23275A, PE labeled). The only problem with these conjugates from BD-Pharmingen is the amount of free fluorochrome (eg. PE) in the prep. Don't hesitate to contact me. Best regards, Joost Schuitemaker _________________________________________ J.H.N. Schuitemaker Research Technician Dept. Cell Biology & Histology Cellular Immunology Group P.O.box 22700 1100 DE Amsterdam The Netherlands Tel. +31 (0)20 5664960 Fax. +31 (0)20 8725456 E-mail privé: J.H.N.Schuitemaker@consunet.nl -----Original Message----- From: Cheryl Smith [mailto:cheryla.smith@utoronto.ca] Sent: maandag 21 februari 2000 18:36 To: cyto-inbox Subject: dilution of antibody Hello out there, One of the users of our facility has a concern regarding the advisability of diluting a particular antibody. Since these reagents are quite expensive she doesn't want to go ahead assuming that all will be well. Please respond to her following question directly at: sandra.aswald@utoronto.ca Thanks I am planning to use the antibody: anti-human IL-12 (p40/p70) mouse IgG FITC; cat.# 18994A from Pharmingen. It should be used at aprox. 0.5ug per 10 million cells, and comes as 0.1mg in a total volume of 200ul. I was wondering a) use only 1ul undiluted AB per 10 million cells b) dilute 1:10 and use 10ul Question: .Since I will be doing intracellular FACS with permeabilized cells, is it O.K. to dilute the antibody? .If I can dilute it, is there an alternative to using the Pharmingen Perm/Wash (TM) buffer? This is the only thing that Pharmingen suggests diluting their anti-cytokine AB's with. Thanks for your help Sandra
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