Hi, Temperature of incubation with Abs to cell surface antigens Simon Monard gives the anti-capping reason and it is an excellent one. Maybe those who prefer ambient temperature (whatever that may be in various parts of the world) get away with it because some cell surface molecules are very much less likely to cap than others. Surface Ig on B cells caps in the twinkling of an eye, as the elderly among us will recall from the early-70s controversy about whether it was there at all. This argument was resolved by incubating lymphocyte suspensions at 4 degrees during staining with anti-Ig and keeping them cold. There is an additional fundamental reason, which concerns Ab affinity (strictly, avidity since practically everyone uses divalent antibody). Where the values have been published, the off-rate constants for defined antibodies such as monoclonals bound to cell surface molecules are about ten-fold less at 4 degrees than at 25 degrees. (There aren't many such determinations, so I hope I'm not over-generalising). For an example that I'm familiar with, the half-time for dissociation of Fab of MRC OX7 (anti-Thy1.1; sorry - anti-CD90) increased from 700 seconds at ambient (actually 18 degrees - this is England!) to 7000 seconds at 4 degrees. (Fab)'2 of W3/25 (anti-rat CD4) is another example. The message is to keep the cells cold (4 degrees) not only during incubation but also during washing afterwards right up to the addition of fixative. People get away with ambient because (1) there may be a compensating acceleration of on-rates at the higher temperature (2) they think they don't mind losing a bit of signal (what you never see you never miss) (3) some monoclonals may have sufficient oomph to keep hanging in there even in heat waves. If you want to read more on this, go to the chapter on "Kinetics of antibody reactions and the analysis of cell surface antigens" by Don Mason and the late Alan Williams. It's Chap 38 in volume 1 of Handbook of Experimental Immunology, ed Weir DM et al, Blackwells Scientific Publications, 4th edition (1986) ISBN 0-632-00975-6 (the off-rate data I have quoted are in a Table there). When you read this, you will also come to challenge the almost universal advice from commercial staining protocols about how much Ab to add during cell labelling reactions (e.g. "one microlitre per 5 million cells" or somesuch instruction). What matters is the absolute concentration of antibody molecules (in relation to the antibody affinity and the proposed staining time), not the ratio of antibody to the number of cells in the incubation mix. In saturating antibody conditions, only a tiny amount of the staining antibody latches on to the cell surface during an incubation (much much less than 5% - the rest goes down the sink). So upping the cell number, within reason, makes very little difference. It *does* matter how much reaction volume you dilute your staining antibody into. But all this is a different point. As usual, when you're setting up the protocol for your favourite antibody, invest a little time and do the experiment on the temp; Ab concn; staining time; cell number! Don Mason has kindly seen this email before it's despatched to the four corners by the e-pigeons, and is content with what it says. He adds: "Ab on-rates can also differ widely [from one Ab to another]. W3/25 is x14 faster than W3/13 [anti-rat CD43], at least at 4 degrees C. Consequently one may have to use some MAbs at higher concentration than others, just to compensate for the low on- rate. In fact we did not appreciate the slow on-rate for W3/13 when we started the study that you mention. The finding that it was so slow resolved the apparent contradiction that the site number for W3/13 on lymphocytes was higher than that for W3/25 by absorption assays but lower by binding assays....". He reinforces the advice to do a few labelling tests at first. Simon Hunt ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Dr Simon V. Hunt, Lecturer in Immunology, University of Oxford Mail to: Dunn School of Pathology, South Parks Road, OXFORD OX1 3RE, U.K. Phone:+44/0 1865 275575 (office/voice mail) 275574 (lab). Fax: 275515 (general office) 285744 (lab personal) http://dunn1.path.ox.ac.uk/~svhunt/labhomepage/SVHunt_WebHomeFeb2001v1.htm Fellow in Immunology and Tutor in Medicine, Keble College Mail to: Keble College, OXFORD OX1 3PG, U.K. Phone:+44/0 1865 272749 (voicemail). Fax: 272705 http://senior.keble.ox.ac.uk/fellows/ ----- Original Message ----- From: "Simon Monard" <smonard@trudeauinstitute.org> To: cyto-inbox Sent: Monday, March 12, 2001 9:18 PM Subject: Re: Hi FLOWers, > > Hi > > People always used to incubate on ice for 30-60 minutes to try to prevent capping, also > to create a convenient time to have lunch or extended coffee and smoking breaks. Nowdays > you can add a tad of Sodium azide to prevent capping. > > Simon > > Simon Monard > FACS Lab Manager > Trudeau Institute > Saranac Lake > NY12983 > > Ph 518 891 3080 X352 > > > >>> "Heather Medbury" <Heather_Medbury@wsahs.nsw.gov.au> - 3/11/2001 5:50PM >>> > > Hi FLOWers, > > About 5 years ago when I was analysing lymphocytes, we used to have the cell preparations > at 4 C for labelling with the antibody. > The work I do now on whole blood, the cells are labelled at room temperature. > > Why do people label at 4 C when it works perfectly well at room temp? > > Heather........
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