MHC Class II (I-A/I-E) Rat Monoclonal Antibody

1. Take 100 μl peripheral blood anticoagulated by EDTA and add to the bottom of 5 ml tube. 2. Add appropriate amount of antibody to the bottom of flow tube mixing with the whole blood; incubate for 30 minutes at room temperature. 3. Add 2 ml RBC lysis buffer; incubate for 10 minutes after mixing; dissolve red blood cells. 4. Sample tube is set to 1000 rpm centrifugation for 5 minutes; discard the supernatant. 5. Add 2 ml PBS wash buffer to resuspend the cells; then 1000 rpm centrifugation for 5 minutes; discard the supernatant. 6. Add appropriate amount of fluorescent-labeled anti-mouse IgGs and incubate for 20 minutes away from light at room temperature. 7. Add 2 ml PBS wash buffer to resuspend the cells; then 1000 rpm centrifugation for 5 minutes; discard the supernatant. 8. Add 0.5 ml PBS wash buffer to resuspend the cells and detect by flow cytometry (sample should be determined on the day on the machine and can also be added fixation overnight at 4 °C then measured) .