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
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