Date: Wed Sep 13 2006 - 05:43:28 EDT
Hi V., could you please elaborate on that? This is the type of discussion I continuously see when critically evaluating data re: cell membrane binding versus diffuse internalization. True sceptics will dismiss classical fluorescence microscopy images showing cytoplasmic staining (diffuse or punctate), arguing you really need confocal to decide whether you're seeing surface stain versus intracellular - and I do see their point. The way I understand the Amnis device works, confocal is really not an option. Seeing the individual cell's staining pattern may therefore be nice to have above and beyond the dotplots/histo's and whatnot outputs as from a conventional cytometer, but shouldn't be used as a definitive argument for internalization. I would therefore argue you can use the Amnis to gather more data from your cells, but for this particular application having the macrofocal image shouldn't make the decisive difference. Doing the "stripped cells" experiment, using fluor-specific secundaries, quenching and/or pH sensitive dyes will therefore be as necessary as when using a non-imaging device. Just my two cents, and purely based on my limited knowledge of the Amnis of course. If it does do confocal as well - hey guys, if you read this, write it in block capitals on all your flyers and put me down for one machine to start with! ;-) Guy Next generation therapeutic <http://www.ablynx.com/> antibodies Guy Hermans, PhD Senior Scientist Ablynx NV Technologiepark 4 B-9052 Zwijnaarde Belgium guy.hermans@ablynx.com tel: fax: mobile: +32 (0)9 261 06 57 +32 (0)9 261 06 27 +32 (0)486 788 551 <https://www.plaxo.com/add_me?u=30065269879&v0=994426&k0=2009290972> Add me to your address book... <http://www.plaxo.com/signature> Want a signature like this? -----Original Message----- From: rozenkov@netscape.net [mailto:rozenkov@netscape.net] Sent: Tuesday, September 12, 2006 7:12 AM To: cyto-inbox Subject: Re: receptor internalisation analysis An ImageStream flow cytometer from Amnis could do this with no additional procedures... Regards. Vladislav Rozenkov -----Original Message----- From: julie.bertout@ibl.fr To: cyto-inbox Subject: receptor internalisation analysis Hi, I have a researcher interested in quantitating receptor internalisation after different treatments. Here is the way she used to do it : - label receptors of interest with primary antibody and secondary antibody - do the treatment she wants to test - remove label from non-internalised receptor with a slightly acid treatment (so only internalised receptors will still be fluorescent) - analyse her samples fluorescence to see a difference. the problem is that, as antibody/receptor afinity is different from one receptor to another, she can't do her positive and negative controls... for example, the receptor which she used as control (which internalisation shouldn't change with/without treatment) has such a high affinity with its antibody that she can't remove it with the acid treatment. So I would like to know if there is any way to quench fluorescence for the receptor that are not internalised or if anybody has a protocol to quantitate internalised receptor? I thought of comparing non-permeabilised vs permeabilised samples but PFA fixation permeabilise cells a little, doesn't it? Thank you for your answers, Julie Bertout cytometry lab Institut Pasteur de Lille 1 rue du professeur Calmette 59800 Lille France _____ <http://pr.atwola.com/promoclk/100122638x1081283466x1074645346/aol?redir =http%3A%2F%2Fwww%2Eaim%2Ecom%2Ffun%2Fmail%2F> Check Out the new free AIM(R) Mail -- 2 GB of storage and industry-leading spam and email virus protection. ----------------------------------------------------------------------- THIS E-MAIL MESSAGE IS INTENDED ONLY FOR THE USE OF THE INDIVIDUAL OR ENTITY TO WHICH IT IS ADDRESSED AND MAY CONTAIN INFORMATION THAT IS PRIVILEGED, CONFIDENTIAL AND EXEMPT FROM DISCLOSURE. If the reader of this E-mail message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please notify us immediately at ablynx@ablynx.com. Thank you for your co-operation. "NANOBODY" and "NANOCLONE" are registered trademarks of Ablynx N.V. -----------------------------------------------------------------------
