Instructions For UseFor the detection of cell cycle antigens such as Ki-67, PCNA and BrdU, methanol modification is recommended - see protocol #F5.
1. Prepare cells in the appropriate manner. Adjust cell suspension to a concentration of 1 x 107
cells/ml in PBS/BSA. Whole blood samples may also be used. Bio-Rad recommend the use of EDTA anti-coagulant in these circumstances, although satisfactory results may be obtained using heparin or acid-citrate dextrose.
2. Add 100ul of cell suspension to the appropriate number of test tubes.
If required, perform staining of cell surface antigens at this stage. Following staining for the recommended period, wash cells once in PBS/BSA and discard supernatant.
3. Add 100ul of Reagent A (fixation medium, stored at room temperature).
4. Incubate for 15 minutes at room temperature.
5. Add 3ml PBS/BSA and centrifuge for 5 minutes at 300 x g. Remove supernatant.
6. Resuspend cells in 100ul of Reagent B (Permeabilization Medium).
7. Immediately add recommended volume of the appropriate directly conjugated antibody. Vortex and incubate for 30 minutes at room temperature.
If using an unconjugated primary antibody, wash in 3ml of PBS/BSA (as per step 5) and then repeat step 7 using an appropriate secondary antibody. There is no requirement to add further Leucoperm™
8. Wash once in PBS/BSA. Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.25ml of 0.5% formaldehyde and store them at 2-8o
C in the dark. Analyse fixed cells within 24 hours.