IGEM:Harvard/2007/Protocols/Indirect Magnetic Labeling Protocol

1. After preparation of single-cell suspension, count cells, and centrifuge cell sample.
2. Resuspend cell pellet and stain with the primary antibody according to the manufacturer's recommendations.
   1. 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.
3. 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.
4. 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.
5. Repeat washing step
6. 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.
7. Mix well and refrigerate for 15 minutes (4C)
   1. (Optional) When using unconjugated or biotinylated primary antibodies, cells can be fluorescently stained with a fluorochrome-conjugated antibody at this step.
8. Wash cells by adding 1-2 mL of buffer per 10^7 cells and centrifuge at 300xg for 10 minutes. Aspirate supernatant completely.
9. Resuspend up to 10^8 cells in 500 ul buffer.

1. • Proceed to Magnetic Separation