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Mouse CD8 T Cell Isolation Kit (Column-Based)

Mouse CD8 T Cell Isolation Kit (Column-Based)

Catalog Number: S0K1007 Reactivity: Mouse Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Mouse CD8 T Cell Biotin-Antibody Cocktail

1.5 mL Streptavidin Beads
Capacity

Suitable for a total cell count of 1×10⁹, and can perform up to 100 sorting runs.
Principle of Separation

This kit employs a column-based negative selection method to isolate CD8 T cells from mouse spleens. In this negative selection process, non-CD8 T cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-nanomagnetic beads. As a result, the non-target cells become bound to the beads via the antibody‑bead complex, while the target CD8 T cells remain unlabeled. The cell suspension is subsequently loaded onto the separation column; under the influence of a magnetic field, the magnetically labeled non-target cells are retained within the column, whereas the unlabeled CD8 T cells elute by gravity into the collection tube. The resulting supernatant represents the CD8 T cell‑enriched fraction after removal of the non-target cells.
Reactivity Mouse
Magnetic Bead Size 20 nm
Separation Method Column-Based
Selection Strategy Negative Selection
Stability & Storage

Store protected from light at 2–8 °C; do not freeze.

Background

Mouse CD8⁺ T cells are the core cytotoxic effector cells of the murine adaptive immune system. They primarily mature in the thymus and are widely distributed throughout peripheral immune organs and tissues. These cells serve as a critical defense against intracellular pathogens (such as viruses and intracellular bacteria) and the clearance of abnormal cells (such as tumor cells). They also participate in the formation of immunological memory and the regulation of immune responses, making them of great significance in immunology research and experimental applications. After isolation, the cells remain unmarked by surface antibodies and retain their native state, enabling subsequent in vitro activation, differentiation, and functional studies.

Protocol

Note: This kit does not include separation buffer; you need to prepare it yourself. The corresponding Stater catalog number isS0D3018

TakingL Separation Columnas an example

Steps

Operating instructions

Dosage and time

Cell processing and labeling

1

Take the mouse spleen, grind it on a 40 μm sterile filter mesh to prepare a single-cell suspension of the spleen, perform red blood cell lysis, and then count the cells.

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 CD8 ratio.

1×107cells/100 μL

3

Add 10 μL of Mouse CD8 T cell Biotin Antibody Cocktail to the sample and gently mix.

Note:In this step, the mixture can be gently pipetted 2–3 times to homogenize;

10 μL/100 μL

4

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

Incubate at room temperature for 10 min

5

Add MagSep Separation Buffer equal to ten times the cell volume to wash the cells, centrifuge at 400g for 7 min, discard the supernatant, and resuspend to the original volume;

Centrifuge at 400g for 7 min

6

Mix wellStreptavidin Beads, add 15 μLStreptavidin Beadsto the sample;

15 μL/100 μL

7

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

Incubate at room temperature for 5 min

Cell sorting

8

In the column separation magnet, installL Separation Column, add 3 mL of MagSep Separation Buffer to 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 500 μL; if insufficient, make up the volume with MagSep Separation Buffer. The cells that drip down under gravity are the target cells.

Collect the target cells

10

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 maximum number of labeled cells in the S Separation Column is1×107;The maximum number of labeled cells in the L Separation Column is1×108;

2. If the viability of the sample is low, it is recommended to use the Dead Cell Removal Kit (Dead Cell Removal Set, (RUO)#S0K0009) to remove dead cells.


Picture

Validation Data

CD8+ T cells were enriched from mouse splenocytes using a CD8+ T cell positive-selection kit. Cells were stained with CD8-Alexa Fluor® 647 (SDT Cat: S0B5895) and CD4-Alexa Fluor® 488 (SDT Cat: S0B8030) fluorescent antibodies, and live cells (Live+) were gated for analysis to determine the proportion of CD8+ T cells in the sorted fraction. Flow cytometric analysis revealed that the purity of CD8+ T cells after this sorting was 98.5%.