Dear Colleagues, As requested, these are the responses I received for my question on CTLA-4 expression. We are in the process of trying these suggestions. Once again, many thanks to those who responded to these questions. Best regards Rohan Ameratunga Auckland Hospital NEW ZEALAND The original question was; Dear Colleagues, I would be grateful for advice on CTLA4 staining. We are attempting to stain cell surface CTLA4 as marker of solid organ rejection in peripheral blood lymphocytes. None of liver or kidney transplant samples have been positive using the Coulter immunotech antibody. We are not sure if this is a technical problem or whether the antigen is not expressed in the periphery. 1. What in vitro stimulation methods should be used for positive controls? How long should cells be stimulated before we detect CTLA4? 2. How useful is intracellular staining for this antigen? Thank you in advance Best regards Rohan Ameratunga Auckland Hospital NEW ZEALAND We only found CTLA4 to be surface-expressed on some donors, at some times. It is highly expressed intracellularly after stimulation with PHA/IL2. Jeff Carrell Human Genome Sciences Try to use either 1-2% PHA or 1% Con A to stimulate the cells for 3 days before staining. Good luck. This was from Dr Tang at Pharmingen We use Concanavalin A treated (3 days) PBMC to get the CTLA-4 antigen to express. It may give you about 40 - 50% of the population showing reactivity. In tissue, we have used frozen thymus. it shows binding on the medulla of thymus. There is a recent paper: Castan, J., U. Klauenberg, P. Kalmar, et al. 1998. Expression of CTLA-4 (CD152) on human medullary CD4+ thymocytes. Med. Microbiol. Immunol 187: 49. I hope this helps Good Luck Enoc Hollemweguer PharMingen Product Manager There was a pamphlet by Pharmingen telling that intracellular staining with the cytokine staining protocol enhances the staining significantly. So you best check there. Otherwise I would have to investigate in my files which may take some time. Kind regards, Dirk Dirk Behringer (behringer@mm11.ukl.uni-freiburg.de) Medizin I, Universit„tsklinik Freiburg, Hugstetterstr. 55, 79106 Freiburg Tel: 0761-270 3505; FAX: 0761-2703684 I've been studying CTLA-4 on peripheral blood lymphocytes and have had good results with the coulter immunotech CTLA-4 antibody after 48 hours stimulation with the anti-CD3 antigen OKT3 at 37C in RPMI + 10% AB serum. I use 5 microlitres OKT3 in 500 ml PBS for each well (1 x 106 cells). Hope this is useful Sophie Scrivener
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