MPO, CD22 抗体

Protocol for sample preparation and intracellular staining. 1. Pipette 50 µl of cell suspension to be analysed (up to 10^6 cells) into each tube. 2. For each sample, add an appropriate volume of conjugated antibody (20 µl) directed to the cell surface antigen of interest and the appropriate isotype control. Incubate for 15 minutes in the dark at room temperature. (This step is only necessary is you want to perform a direct immunofluorescence staining for a cell surface antigen) 3. Add 100 µL of Fix & Perm Reagent A, (Fixative), to each tube. Mix gently. 4. Incubate for 15 minutes at room temperature. 5. Wash once in 2 ml PBS Working Solution 1X. 6. Centrifuge for 5 minutes at 300 xg, then aspirate supernatant, leaving approximately 50 µl of fluid and vortex to ensure that the cell pellet are in suspension. 7. Add 100 µL of Fix & Perm Reagent B (Permeabilization), to each tube. Add the appropriate volume of conjugated intracellular antibody specific for the intracellular antigen and the appropriate isotype control. 8. Incubate for 15 minutes in the dark at room temperature. 9. Wash once in 2 ml PBS Working Solution 1X. 10. Centrifuge for 5 minutes at 300 xg, then aspirate supernatant, leaving approximately 50 µl of fluid and vortex to ensure that the cell pellet are in suspension. 11. Resuspend the cell pellet in 0,5 ml of 1% paraformaldehyde solution or an appropriate fluid for flow cytometric use, and store in the dark at 2-8 °C. Fixed cells should be analyzed within 24 hours.