Flow Cytometry Procedure

We use the monoclonal antibodies at 15 μg/ml, 50 μl/10⁶ cells in 100 μl final volume for single labeling. For multicolor labeling, the volume is 150 μl for two color and 200 μl for three color.  We incubate for 15 minutes on ice, wash 3X, then add the appropriate titered second-step reagents and incubate for another 15 minutes.  We then wash 2X and fix in 2% PBS buffered formaldehyde.  The incubation medium contains gamma-globulin-free horse serum or other protein that does not contain bovine immunoglobulin. Regular BSA contains some immunoglobulin.  When working with pigs, it won't make any difference unless PIG45A2 (anti-IgM) is being used.  PIG45A2 recognizes a conserved determinant on bovine and porcine IgM.

Our sources for secondary antibodies, polyclonal and isotype-specific, human absorbed, are Caltag Laboratories and Southern Biotechnology Associates, Inc. The reagents have to be titered for use.  The fluorescein- and PE-conjugated reagents are available from both companies.  PE-Cy5, anti-IgG1 (TRICOLOR), is only available from Caltag Laboratories.  I have not used other sources of second-step reagents.

This procedure is supplied by Dr. Bill Davis at Washington State University.