Mouse Lineage Cell Depletion Kit (Column-Based)

the steps

and operating instructions

for dosage and timing are as follows:

Cell processing and labeling

1

Collect mouse bone marrow, prepare a single-cell suspension, and perform cell counting;

Note:Mouse bone marrow must undergo red blood cell lysis using Red Blood Cell Lysis Buffer (without fixative, 1X) (Cat: S0D9002); it is recommended to count cells after staining with AO/PI;

Sample preparation

2

Resuspend 1 × 10⁷live cells in 100 μL of pre-cooledMagSep Separation Buffer(Cat: S0D3018);

Note:Adjust reagent volumes proportionally according to sample volume; retain a pre-sorting sample to determine the Lin- cell percentage;

1 × 10⁷cells/100 μL

3

Add 10 μL of the Mouse Lineage Cell Depletion Biotin-Antibody Cocktail to the sample and gently mix.

Note:In this step, the mixture can be gently mixed by pipetting up and down 2–3 times.

10 μL/100 μL

4

Incubate the antibodies and cells in the cocktail at 2–8°C for 10 minutes.

Incubate at 2–8°C for 10 min.

5

Add 10 times the cell volume of pre-cooledMagSep Separation BufferWash the cells, centrifuge at 400g for 7 minutes, discard the supernatant, and resuspend to the original volume.

Centrifuge at 400g for 7 minutes.

6

Mix the Streptavidin NanoBeads thoroughly, then add 10 μL of Streptavidin NanoBeads to the sample.

10 μL/100 μL

7

Gently mix the magnetic beads and cells, and incubate at 2–8°C for 5 minutes.

Note:In this step, gently pipette up and down 2-3 times to mix thoroughly;

Incubate at 2-8℃ for 5 min

Cell sorting

8

In the column-based magnetic separator, install the L Separation Column and add 3 mLMagSep Separation Bufferto rinse the separation column;

Rinse the separation column

9

Add the cells to the separation column and collect the target cells using a collection tube;

Note:The cell volume should be at least 1 mL; if insufficient, useMagSep Separation Bufferto make up the volume; the cells that drip down under gravity are the target cells;

Collect the target cells

10

Add 3 mL ofMagSep Separation Bufferthree times, collecting the target cells each time;

Note:This step elutes the unlabeled target cells from the separation column, increasing the yield.

Collect the target cells

Notes: 1. Pay attention to the maximum capacity of the separation column; the S Separation Column can handle up to 1×10⁷ labeled cells; the L Separation Column can handle up to 1×10⁸ labeled cells;

2. Throughout the entire process, pay close attention to temperature control; keep the sorting buffer pre-cooled at all times and avoid leaving it at room temperature for extended periods.