Getting antibody into viable cells

ray hester rhester at jaguar1.usouthal.edu
Wed Mar 27 10:06:49 EST 2002



Greetings,

Below is one protocol suggested for the introduction of antibodies into
viable cells that wasn't posted to the entire list:

............................................................................
............
might try sterile saponin solutions


Saponin Method: Permeabilization REAGENT for Paraffin sections &
Itracellular Cytokine staining

Dulbecco's PBS (without Mg2+ or CA2+)
1% heat inactivated FCS (or BSA-do not use animal serum with
 Biotin detection systems due to endogenous biotin levels)
0.1% w/v sodium azide
0.1% w/v saponin (Sigma cat#S-7900)
Adjust pH buffer to 7.4-7.6 and filter

see ref:
1)Sander, B., J. Andersson and U. Andersson, 1991:Assessment of
cytokines by immunofluorsecence and the paraformaldehyde-saponin
procedure:Immunol Rev 119:65-93

2) Anderson, U. and J. Andersson 1994. Immunolabelling of cytokine
producing cells in tissues and suspension. In Cytokine Producing Cells
eds. D. Fradelizie and D. Emelie. INSERM, Paris. 32-49.


for intracellular cytokines, stop golgi process with with either
monesin (Sigma cat#M-5273) or Brefeldin A (Sigma cat#B-7551)

-ref#Nature 373:255-257

-Prussin C and Metcalfe DD.  Detection of Intracytoplasmic Cytokine
Using Flow Cytometry and Directly Conjugated anti-Cytokine Antibodies.
Journal of Immunological Methods, 188, 117-128 (1995).


-J. Immunol Meth 159:197-207

see antibodies at: http://www.researchd.com/absort.htm



Sincerely,
Research Diagnostics Inc
Pleasant Hill Road
Flanders NJ 07836
phone 973-584-7093
fax 973-584-0210
email: researchd at aol.com
web: http://www.researchd.com





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