ABCG2/CD338 Antibody (3G8):
Flow Cytometry Protocol
Solutions and Reagents:
1X PBS
Blocking buffer: 0.5% BSA in 1X PBS
Filter buffer: 0.1%Triton-X100 and Blocking buffer
Ice cold 4% paraformaldehyde (1% solution -optional for storing samples)
Fluorescently-conjugated secondary antibody (various forms)
Protocol
2.1. Collect 1x10^6 cells/sample.
2.2. Wash cells once with blocking buffer.
2.3. Fix cells with 4% paraformaldehyde and incubate at 4C for 30 min.
2.4 Permeabilize cells: Add 0.5 ml filter buffer(0.1%Triton-X100 and Blocking buffer) and incubate at 0 degree C for 15 min.
2.4. Wash cells once with blocking buffer.
2.5. Add 0.5 ml filter buffer and incubate at 0C for 15 min.
2.6. Wash cells twice with blocking buffer.
2.7. Incubate cells in blocking buffer for 10 min at room temperature.
2.8. Add primary antibody at the appropriate dilution and incubate for 30 min at room temperature.
2.9. Wash twice with blocking buffer and incubate with fluorescently-conjugated secondary antibody for 30 min at room temperature.
2.10. Wash cells twice with blocking buffer.
2.11. Re-suspend cells in 1X PBS and analyze on flow cytometry. Samples can be kept in 1% paraformaldehyde at 4C overnight.
Advice: Keep the cells in the dark on ice or at 4C in a fridge until your scheduled time for analysis. Analyze the cells on the flow cytometer as soon as possible.
