1. | Use the cell suspension obtained in Table 1. |
2. | Add 20 μL of FcR blocking reagent. |
3. | Mix well and incubate at 4°C for 10 minutes. |
4. | Add 30 μL of anti-prominin-1 microbeads antibodies. |
5. | Mix well and incubate at 4°C for 10 minutes. |
6. | Add 10 μL of anti-prominin-1 APC antibodies. |
7. | Mix well and incubate at 4°C for 5 minutes. |
8. | Wash cells by adding 10 mL of dissociating buffer. |
9. | Centrifuge at 500 g for 10 minutes. |
10. | Aspirate the supernatant completely. |
11. | Expand the cell pellet in 500 μL of dissociation buffer. |
12. | Apply the solution to an LS column primed with 3 mL of buffer. |
13. | Rinse the column with 3×3 mL of buffer, keeping the LS column away from the magnet. |
14. | Collect the flow through solution and centrifuge at 500 g for 10 minutes. |
15 | Discard the supernatant and add 500 μL of buffer. |
16. | Insert anew LS columns into an MACS separator magnet and prime it with3 mL of buffer. |
17. | Apply the cell suspension obtained in 15. |
18. | Wash the column with 3×3 mL of buffer. |
19. | Remove the column from the separator and place it on a suitable collection tube. |
20. | Flush the magnetically labeled cells with 5 mL of dissociation buffer. |