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Note: This method provides a general
procedure for use with the majority of Serotec reagents. In some cases
specific recommendations are provided on product datasheets, and these
methods should always be used in conjunction with product and batch
specific information provided with each vial. Please note that a certain
level of technical skill and immunological knowledge is required for the
successful design and implementation of these techniques - these are
guidelines only and may need to be adjusted for particular applications.
1. Add 100ml of cells to each tube. 2. Add 100ml of Leucoperm™ reagent A (Cat. No. BUF09), and incubate for 3 minutes at room temperature. 3. Whilst vortexing gently add 2-4ml of cold
(0-4oC) absolute methanol. 4. Incubate for an additional 10 minutes at
0-4oC. 5. Centrifuge for 5 minutes at 350g, and wash
once with PBS containing 1% BSA. 6. To the cell pellet add 100ml of Leucoperm™ reagent B, and add appropriate volume of FITC conjugated antibody. 7. Vortex briefly at low speed, and incubate
for 30 minutes. 8. Wash once in PBS/1% BSA and analyse by flow
cytometry. N.B. This protocol is not recommended for the simultaneous staining
of cell surface antigens using RPE conjugated
antibodies.
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