I also agree with Greg Hodge; I used PMA and iono for a min. of 12 hours; 18 hours being good. 4-6 hours may be good for human, but doesn't work for rhesus (that was what I used anyway) and I also ficolled the blood. In addition, I used Pharmingen antibodies. As I remmeber, the antibodies I looked at were IFN-g, IL-4, IL-6, IL-8 and GM-CSF. I "seem" to remember having gotton up to 40% positivity for IL-4, but would have to dig out the data to be sure. P. Echeagaray Life Technologies, a Division of Invitrogen pecheaga@lifetech.com >From: andrew.head@covance.com >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> >Subject: IL-4/5 >Date: Tue, 07 Nov 2000 16:14:56 -0500 > > > Dear Flowers > My original question was: Does anyone have any experience of typing > Th1 and Th2 cells in cynomolgus macaque lymphocytes using IL-4 and > IL-5? > Below is a summary of answers I received > Helen Horton from Wisconsin Regional Primate Research Centre. In >order > to test for IL-4/5 Try using the protocol supplied by Pharmingen with > the IL-4mAb,to in vitro switch the monkey cells. > Ursula Banning- We stain Human lymphs for IFN-g + IL-4 using PMA and > ionomycin for 4 hours and works well. > Becky Kelly from Wisconsin University of medicine- In human lymphs we > see a small number of IL-5+ CD4+ cells(~1%).This is true of PBMC >preps > and bronchial lavage cells. Subjects who have undergone airway > allergen challenge and cultured cells secrete large amounts of > IL-5(ng/ml) > Calman Prussin from NIAID suggested getting a pos control(Pharmingen > sell some)Also may get more easily observed staining if you can gate > on a memory cell pop such as CD27 neg Tcells.Essentially all IL-4 and > 5 staining is limited to that subset which is only 5-10% of all CD4+ > cells. > Julie Turner suggested for a pos control- Incubate cells with anti >CD3 > in presence of IL-4 and anti IL-12(to prime Th2 cells). After a few > days hit with PMA again,Th2 cells should proliferate. > Denise Croix from Dept of mol genetics,Pittsburgh suggested that > Ficoll separation of PBMC's may be a problem with Cynos.Try using > Percoll separation.Also PDBu(Sigma P1269) gives a better response >than > PMA in Rhesus > Greg Hodge said that although most literature suggests 4-6 hrs.he > personally finds 16-18 hours gives a greater % of cells producing > IL-4/5 although you get loss of CD4,increased cell death and >increased > autoflouresence. He stains CD8 fitc/IL-4 PE/CD3 pc5 and assumes > CD3+/CD8- cells are true CD4+ cells.Also A549 epithelial cell line is > a good pos control producing ~ 15% IL-4 at 16 hour timepoint. > Also a couple of references > Prussin,C and D.D Metcalfe(1995). J Immunol Methods 188:117-28 > Detection of intracytoplasmic cytokine > Rostaing,L et al.Cytometry(1999) 35:318-328 Kinetics of > intracytoplasmic Th1 and Th2.. > Hope this helps > Andy > > > >----------------------------------------------------- >Confidentiality Notice: This e-mail transmission >may contain confidential or legally privileged >information that is intended only for the individual >or entity named in the e-mail address. If you are not >the intended recipient, you are hereby notified that >any disclosure, copying, distribution, or reliance >upon the contents of this e-mail is strictly prohibited. > >If you have received this e-mail transmission in error, >please reply to the sender, so that we can arrange >for proper delivery, and then please delete the message >from your inbox. Thank you. _________________________________________________________________________ Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com. Share information about yourself, create your own public profile at http://profiles.msn.com.
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