IGEM:Harvard/2007/Protocols/Indirect Magnetic Labeling Protocol
- After preparation of single-cell suspension, count cells, and centrifuge cell sample.
- Resuspend cell pellet and stain with the primary antibody according to the manufacturer's recommendations.
- For MACS Fluorochrome-conjugated or Biotinylated Antibodies, typically resuspend up to 10^7 total cells in 100 ul of buffer and add 10 ul of the respective antibodies.
- Mix well and refrigerate for 5-10 minutes or according to the manufacturer's recommendations. ##If fluorochrome-conjugated antibodies are used, incubate in the dark.
- Wash cells to remove unbound primary antibody by adding 1-2 mL of buffer per 10^7 cells and centrifuge at 300xg for 10 minutes.
- Repeat washing step
- Aspirate supernatant completely. Resuspend cell pellet in 80 ul of buffer per 10^7 total cells and add 20 ul of indirect MicroBeads per 10^7 total cells.
- Mix well and refrigerate for 15 minutes (4C)
- (Optional) When using unconjugated or biotinylated primary antibodies, cells can be fluorescently stained with a fluorochrome-conjugated antibody at this step.
- Wash cells by adding 1-2 mL of buffer per 10^7 cells and centrifuge at 300xg for 10 minutes. Aspirate supernatant completely.
- Resuspend up to 10^8 cells in 500 ul buffer.