A buffy coat or another mixed cell suspension is mixed with antibody-conjugated microbeads, diluted and placed in a magnetic separation column. Cells bound to the microbeads stick to the side walls of the column, while cells not bound to the magnetic beads flow through the column. By removing the separation column from the magnetic field, the cells bound to the microbeads are released from the column and collected from the column with a pin plug.

Operation method

Scheme 15.2 Magnetic Activated Cell Sorting (MACS)

Principle

A buffy coat or another mixed cell suspension is mixed with antibody-conjugated microbeads, diluted and placed in a magnetic separation column. Cells bound to the microbeads stick to the side walls of the column, while cells not bound to the magnetic beads flow through the column. By removing the separation column from the magnetic field, the cells bound to the microbeads are released from the column and collected from the column with a pin plug.

Materials and Instruments

Buffers
Magnetic Cell Separators Separation Column Adapters Positive Selection Columns Collection Volume Compliant Collectors Magnetic Microbeads

Move

I. Marking

1. Standard methods for isolation of peripheral blood mononuclear cells or cell suspensions prepared by enzymatic digestion and other methods.

2. Dead cells are removed by Ficoll-Paque.

3. Filter the cells through a 30 μm nylon mesh sieve or filter membrane (Myltenyi, # 414: 07) to remove cell clumps (wet the membrane with buffer before application).

4. Wash cells in buffer (D-PBS, pH 7.2, containing 0.5 % BSA and 2 mmol/L EDTA) and centrifuge.

5. Resuspend centrifuged cells in 80 μl of buffer per ¹⁰⁷ cells (80 μl is the minimum, even if the total number of cells is less than ¹⁰⁷ ).

6. Add 20 μl of bead labeling solution per ¹⁰⁷ cells.

7. Mix well and incubate at 6-12°C for 15 min (for fewer cells, use the same volume of bead labeling solution).

8. Dilute the cells by adding 10-20 times the volume of labeling solution.

9. Centrifuge at 300 g for 10 min.

10. Remove supernatant and resuspend bead-bound cells with 500 μl of buffer per ¹⁰⁸ cells.

Positive Magnetic Separation

11. Place the column into the magnetic zone of a MACS separator (MiniMACS, MidiMACS, VarioMMMACS, or SuperMACS (Miltenyi)).

12. Wash the column: MS ^+/- RS ⁺ 500 μl; LS ^+/- VS ⁺ 3 ml.

13. Add cell suspension to the column: ^MS+/RS+ 500~1000 μl; ^LS+/VS+ 1~10 ml.

14. Allow negative cells to flow through the column.

15. Drench the column with buffer: ^MS+/RS+ 3×500 μl; ^LS+/VS+ 3×3 ml.

16. Remove the column from the separator and place it on the collection tube.

17. Use a pipette to add appropriate amount of buffer to the column ( ^MS+/RS+ 1 ml; ^LS+/VS+ 5 ml) and push the syringe plunger to elute positively labeled cells.

18. Count the cells and adjust the concentration with growth medium.

(a) Primary culture was adjusted to ^{1×105~1×106} /ml and inoculated in culture flasks;

(b) Clonal culture was adjusted to 10~1000 /ml and inoculated in culture dishes.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/