Dear Paula, First I would like to give a tip, a (new) possibility at the Purdue site is the search engine for the E-mail archive at: http://www.cyto.purdue.edu/hmarchiv <http://www.cyto.purdue.edu/hmarchiv> . Here you can simply type a word e.g. Granzyme and it will find all E-mails containing this word. However I do not mind to repeat the E-mail which I have send at the 23th of April, since the antibodies we have raised against Granzyme's (both A and B) recognise the natural protein which seems to be unique in the world. We have clones suitable for flow cytometry (PE- and biotin-conjugated), for histology, for precipitation, blotting and ELISA's. For flow cytometry our CLB department of Clinical Viro-Immunology studied the phenotype of effector T cells, using among others Perforine and Granzyme antibodies. Some publications using our antibodies; Hamann et all, Phenotypic and functional separation of memory and effector human CD8+ T cells, J.Ex.Med 1997, 186, 1407-1418. Wever et all, The CD8+ T-cell subset in peripheral blood from healthy individuals contains activated and apoptosis-prone cells, Immunology, 1998, 93, 383-389. The monoclonal antibody to Perforine was obtained from of Holzel Diagnostika, Koln, Germany and the Granzyme-B antibody conjugated to phycoerytrhin is produced in our own institute by CLB-Reagents and is commercially available, please check following link for world wide distributors: http://www.clb.nl/CLBweb/CLBreag.nsf/8dbb4245016776e8c125646d00357d01/c9ab10 6c688e901bc12564e6002e70c4?OpenDocument <http://www.clb.nl/CLBweb/CLBreag.nsf/8dbb4245016776e8c125646d00357d01/c9ab1 06c688e901bc12564e6002e70c4?OpenDocument> . CLB protocol for membrane and intracellular FACS staining : (By Paul Baars, CLB-KVI) Reagents: PBS PBS 0.5% BSA PBS 0.1% saponin 0.5% BSA Human Pooled Serum (HPS) 4% Paraformaldehyde (PFA) in PBS Procedure (the whole procedure is performed on ice) 1. Wash the cells in a 15 ml tube in PBS 0.5% BSA 2. Suspend the cells in PBS 0.5% BSA (4x106/ml) and add direct conjugated Mab's for the membrane staining 3. Incubate for 30 min 4. Wash 1X with PBS 0.5% BSA 5. Wash 1X with PBS 6. Add 1.5 ml 4% PFA in PBS and incubate for 5 min (stopwatch!!) 7. Wash 1X with PBS 8. Wash 1X with PBS 0.1% saponin 0.5% BSA 9. Suspend the cells in PBS 0.1% saponin 0.5% BSA + 10% HPS 10. Incubate for 20 min 11. Wash 1X with PBS 0.1% saponin 0.5% BSA 12. (From now on this buffer is used until the end of the procedure) 13. Suspend the cells in a x 50ml (a= number of different intracellular staining) and pipette the cells in a 96-well dish 14. Add Mab's to the intracellular antigen and control Mab's (diluted in saponin buffer) 15. Incubate for 30 min 16. Wash 3X 17. Measure the cells on the FACS We know that other permeabilization procedures are also successful for Granzyme staining e.g. the BFA fixation, If you want example histograms please let me know so we can send them to you by separate E-mail. At this time we are building a new web site (the old is still on www.clb.nl <http://www.clb.nl> ) which will contain also examples and protocols. Best regards, John John Voorn CLB Reagents Productmanager Plesmanlaan 125 j_voorn@clb.nl <mailto:j_voorn@clb.nl> 1066CX AMSTERDAM the Netherlands tel +31-(0)20 5123246 fax +31-(0)20 5123570 web site www.clb.nl <www.clb.nl>
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