Anti-biotin Nanobeads

Steps

Operating method

Dosage / Time

Magnetic labeling

1

Count total cell number

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2

Centrifuge the cell suspension at 400×g for 7 minutes, and completely discard the supernatant.

400×g, 7 min

3

Resuspend the cells, and label them with biotinylated CD3 antibody according to the manufacturer's instructions. To obtain optimal cell labeling, it is recommended to test different antibody concentrations. Typically, for labeling 10⁷ mononuclear cells, add 0.1 to 5 μg of antibody. Incubation at 2–8 °C for 15 minutes is usually sufficient.

Note:Ensure thorough mixing of the antibody with the cells by gently pipetting up and down or lightly tapping the bottom of the tube.

Biotinylated antibody labeling

4

For every 10⁷ cells, add 1–2 mL of labeling buffer to wash the cells and remove unbound biotinylated antibodies, then centrifuge at 400×g for 7 minutes. Completely aspirate the supernatant.

Note:The washing step can be repeated

Washing cells

5

Resuspend the cell pellet from every 10⁷ total cells in 90 μL of labeling buffer

Resuspending cells

6

Add 10 μL per 1 × 10⁷ cellsAnti-biotin Nanobeads

Note:When the cell quantity increases, proportionally increase the volume of all reagents accordingly

Add 10 μL per 1 × 10⁷ cells

7

Mix thoroughly, incubate in the dark at 2–8°C for 15 minutes

Incubate for 15 minutes

8

For every 1 × 10⁷ cells, add 1 mL of buffer for washing, centrifuge at 400×g for 7 minutes, and completely aspirate the supernatant

Add 1 mL per 1 × 10⁷ cells

9

Resuspend up to 1×10^8 cells in 500 μL of buffer⁸Cells

Resuspend cells

Magnetic separation

10

Place the S-type separation column into the magnetic field of the corresponding magnetic separator, and rinse the column with 500 μL of buffer

Rinse the separation column

11

Load the cell suspension onto the separation column, and collect the flow-through (unlabeled cells)

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12

Wash the column with buffer three times, 500 μL each time; add the next wash only after the liquid in the column has completely drained.

3 × 500 μL of buffer

13

Combine all flow-through fractions as the negative fraction; remove the separation column from the magnetic field and place it over the collection tube.

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14

Add 1 mL of buffer to the column, then quickly and firmly push down with the provided plunger to elute the magnetically labeled positive cells.

Note:If higher purity is required, repeat the separation once using a new column.

Collect the target cells.

Steps

Operating instructions

Dosage and time

Cell processing and labeling

1

Take the mouse spleen and grind it on a 40 μm sterile filter to prepare a single-cell suspension of the spleen. After red blood cell lysis, proceed with cell counting;

Note:It is recommended to count cells after staining with AO/PI; splenic cells can be centrifuged at 400 g for 7 minutes;

Prepare the sample

2

Resuspend 1×10^7 cells in 100 μL of MagSep Separation Buffer;⁷cells;

Note:Adjust reagents proportionally according to the sample volume; set aside a pre-sorting sample to determine the initial T-cell ratio;

1×10⁷cells/100 μL

3

Add 10 μL of Mouse T cell Biotin Antibody Cocktail to the sample and gently mix;

Note:In this step, gently pipette the mixture 2–3 times to mix thoroughly;

10 μL/100 μL

4

Incubate the antibodies in the cocktail with the cells at room temperature for 10 minutes;

Incubate at room temperature for 10 minutes

5

Mix the Anti-biotin Nano beads well,Beads, and add 20 μL of Anti-biotin Nano beads.BeadsAdd it to the sample;

20 μL/100 μL

6

Gently mix the magnetic beads and cells, and incubate at room temperature for 5 minutes;

Incubate at room temperature for 5 minutes

7

In the column separation magnet, installL Separation Column, and add 3 mL of MagSep Separation Buffer to rinse the separation column;

Rinse the separation column

Cell sorting

8

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

Note:The cell volume should be at least 500 μL; if insufficient, add MagSep Separation Buffer to make up the volume; the cells that drip down under gravity are the target cells.

Collect the target cells

9

Add 3 mL of MagSep Separation Buffer and collect the target cells.

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 a maximum of1×10⁷;the L Separation Column's maximum labeled cell count is1×10⁸;

2.If the sample viability is low, it is recommended to use a dead cell removal kit to eliminate dead cells (Dead Cell Removal Set, (RUO)#S0K0009);