Dear Claudio, In our institute the departement of Clinical Viro-Immunology studied the phenotype of effector T cells, using among others Perforine and Granzyme antibodies. Publication; Hamann et all, J.Ex.Med 1997, 186, 1407-1418. The monoclonal antibody to Perforine was obtained from of Holzel Diagnostika, Koln, Germany and the Granzyme-B antibody is produced in our own institute by CLB-Reagents (commercially available). CLB protocol for membrane and intracellular FACS staining : (By Paul Baars) 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 untill the end of the procedure) 13. Suspend the cells in a x 50ml (a= number of different intracellular stainings) 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 at CLB which you success with the protocol, If you want example histograms please let me know so we can send them by separate E-mail. Best regards, John Voorn CLB Reagents tel: +31-20-5123246 Plesmanlaan 125 fax: +311-20-5123570 1066CX Amsterdam E-mail: J_Voorn@CLB.nl <mailto:J_Voorn@CLB.nl> the Netherlands Site: WWW.CLB.nl <http://WWW.CLB.nl> -----Original Message----- From: claudio.vallan@dkf7.unibe.ch [SMTP:claudio.vallan@dkf7.unibe.ch] Sent: maandag 19 april 1999 10:53 To: cytometry@flowcyt.cyto.purdue.edu Subject: intracellular perforin staining Received: from ns-clb.clb.nl by ln210-2.clb.nl (Lotus SMTP MTA v1.05 (274.9 11-27-1996)) with SMTP id C1256758.005B4F60; Mon, 19 Apr 1999 17:39:22 +0200 Received: from flowcyt.cyto.purdue.edu by ns-clb.clb.nl (AIX 4.1/UCB 5.64/4.03) id AA24492; Mon, 19 Apr 1999 18:47:07 +0200 Received: (from root@localhost) by flowcyt.cyto.purdue.edu (8.9.3/8.9.3) id JAA07571 for cyto-sendout; Mon, 19 Apr 1999 09:51:01 -0500 Received: (from daemon@localhost) by flowcyt.cyto.purdue.edu (8.9.3/8.9.3) id DAA06133 for kelley; Mon, 19 Apr 1999 03:47:12 -0500 Received: from arwen.unibe.ch (arwen.unibe.ch [130.92.9.52]) by flowcyt.cyto.purdue.edu (8.9.3/8.9.3) with SMTP id DAA06129 for <cytometry@flowcyt.cyto.purdue.edu>; Mon, 19 Apr 1999 03:47:10 -0500 Received: from ubecx01 (actually ubecx01.unibe.ch) by arwen with smtpL; Mon, 19 Apr 1999 10:49:15 +0200 Received: from [130.92.148.110] (pathomac110.unibe.ch) by ubecx01.unibe.ch (PMDF V5.1-12 #21734) with ESMTP id <0FAF009REHZX0H@ubecx01.unibe.ch> for cytometry@flowcyt.cyto.purdue.edu; Mon, 19 Apr 1999 10:52:46 +0200 (MET DST) Date: Mon, 19 Apr 1999 10:52:46 +0100 From: Claudio Vallan <claudio.vallan@dkf7.unibe.ch> Subject: intracellular perforin staining X-Sender: vallan@ubecx01.unibe.ch To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> Message-Id: <l03110700b340ac7d1aed@[130.92.148.110]> Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" Does anybody have a protocol for intacellular staining of human perforin that works? I used the Ab from Pharmingen on activated intraepithelial lymphocytes of the small intestine and two different methods for permeabilization: PFA + saponin and OrthoPermeaFix. As positive control I used CTL clones. The Ab gave high background but no specific staining. Thanks for your help!
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