In reply to Heather,

Although I'm not a FLOW person, antibody labelling is dear to the heart of many confocalists!

Some antibodies label significantly better (most in fact) at 4°C. Great care must be taken to not let the solutions dry out when preparing samples for microscopy (place the slide in a humidified container), but this is not an issue for "bulk" labelling for FLOW.

Some antibodies are better labelled at room temperature or 37°C, but this is rare and depends on your antibody.

As a general rule, if your antibody works well then room temperature or 37°C is fine. The advantage is that the time can be a lot shorter (1 to 2 hours). At 4°C the labelling requires more time (it is often convenient to leave overnight). These times refer to labelling fixed cells, live cells can often be labelled for much shorter times - even as low as a few minutes. Low temperature often increases the specific binding and lowers the background, but be warned that 4°C can result in bright spots of fluorescence that may be due to precipitation of the label.

These issues are discussed in more detail in Chapter 11 "Fluorescence Immunolabelling" of my manual "Confocal Microscopy for Biologists".

Good luck, Alan Hibbs.

The manual "Confocal Microscopy for Biologists" is still available from BIOCON.
BIOCON, specialists in confocal microscopy         ABN: 46 196 908 558
7 Walhalla Drive, Ringwood East VIC 3135 Australia      phone: 61 3 9876 9822
Dr. Alan R. Hibbs                                                                        FAX: 61 3 8660 2290	


-----Original Message-----
From:   Heather Medbury [SMTP:[log in to unmask]]
Sent:   Wednesday, March 07, 2001 2:34 PM
To:     [log in to unmask]
Subject:        temperature of facs preps

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

Heather Medbury (PhD)
Department of Surgery
Westmead Hospital
Westmead 2145
61-2 9845 7677
--------------------------------------------------------------
     All experiments are preliminary

------------------------o0)(0o--------------------------------------

  "In his heart a man plans his course
    but the Lord determines his steps"

---------------------------------------------------------------