Melanoma Stem Cell Isolation: A Method to Obtain CD133 Positive and Negative Melanoma Cells Using Magnetic-activated Cell Sorting

1. Magnetic Cell Sorting of CD133⁺ and CD133^– Melanoma Cells Using LS Columns

1. Prepare MACS buffer containing 2 mM EDTA and 5% FCS in PBS. Mix by inverting and sterile-filtrate buffer using a 0.22 μm Steritop-GP Filter Unit. Chill buffer to 4-8 °C and degas before use.
2. Pre-warm the Accutase, PBS, and Quantum 263 medium to 37 °C.
3. Wash 80% confluent cells with PBS. Add an appropriate amount of Accutase and incubate until cells detach from the culture surface. Collect cells in a 50 mL tube using Quantum 263 medium.
4. Determine the cell number and centrifuge the required number of cells for 5 min at 300 x g and 4-8 °C. (According to Miltenyi's protocol, a maximum of 2x 10⁹ cells can be loaded per LS Column. Cell line-specific optimal numbers should be determined and can be considerably lower.) Aspirate supernatant completely.
5. Resuspend a maximum of 1x 10⁸ cells with 350 μL MACS buffer (for higher cell numbers, scale up all following MACS buffer, FcR Blocking Reagent, CD133/1-Biotin, and anti-Biotin MicroBeads volumes accordingly).
6. Add 100 μL FcR Blocking Reagent.
7. Add 50 μL CD133/1-Biotin. Mix well using a vortex and incubate at 4-8 °C for 10 min.
8. Wash cells by adding 10 mL MACS buffer and centrifuge for 5 min at 300 x g and 4-8 °C. Aspirate supernatant completely.
9. Repeat washing step (1.8).
10. Resuspend cell pellet with 400 μL MACS buffer.
11. Add 100 μL anti-Biotin MicroBeads. Mix well using a vortex and incubate at 4-8 °C for 15 min.
12. Meanwhile, prepare the MACS separator for magnetic separation. Attach QuadroMACS to MACS Separator Multi Stand. Insert the required number of LS columns with the column wings to the front in the magnetic field of the QuadroMACS. Place a Pre-Separation Filter (with 30 μm nylon mesh) into each LS column and an appropriate collection tube (15 mL or 50 mL) under each column. Prepare filter and column by rinsing with 3 mL MACS buffer. Discard flow-through.
13. Wash cells by adding 10 mL MACS buffer and centrifuge for 5 min at 300 x g and 4-8 °C. Aspirate supernatant completely.
14. Resuspend cells with 500 μL MACS buffer and apply cell suspension onto the LS column with the Pre-Separation Filter.
15. Wash three times with 3 mL MACS buffer per column. Only add the fresh buffer when the column reservoir is empty.
16. The total collected effluent contains the unlabelled CD133^– cell fraction.
17. Discard the Pre-Separation Filter, remove the column from the separator and place it on a suitable collection tube.
18. Apply 5 mL MACS buffer. Immediately flush out the labeled CD133⁺ cells by firmly pushing the plunger into the column.
19. To increase the purity of the negative fraction, apply cell suspension onto a new equilibrated LS column inserted in the QuadroMACS. The effluent contains the CD133^– fraction.
20. Discard the columns.