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Mouse CD8 T Cell Isolation Kit (Column-Free)

Mouse CD8 T Cell Isolation Kit (Column-Free)

Catalog Number: S0K3004 Reactivity: Mouse Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Mouse CD8 T Cell Biotin-Antibody Cocktail

1.5mL Streptavidin Beads
Capacity

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

In this negative selection process, non- is labeled with a biotin-conjugated antibody.CD8 TCells are then incubated with streptavidin‑coated nanomagnetic beads; non‑target cells are labeled via antibody‑bead complexes, while the target…CD8 TThe cells remain unlabeled. The cell suspension is then transferred to a polystyrene tube and placed within the sorter's magnetic field. Under the influence of the magnetic field, the magnetically labeled non-target cells adhere to the tube wall, while the unlabeledCD8 TThe cells remain in the supernatant. This supernatant is, therefore, the solution from which non-target cells have been removed.CD8 TCell-enriched fraction.
Reactivity Mouse
Magnetic Bead Size 150 nm
Separation Method Column-Free
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 sorting buffer; please prepare it yourself. The corresponding Stater product number isS0D3018

Steps

Operating Instructions

Dosage and Time

1

Collect the mouse spleen, grind it on a 40 μm sterile filter to prepare a single-cell suspension of splenic cells, and perform 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;

Sample Preparation

2

Resuspend 1×10 in 100 μL of MagSep Separation Buffer.7Cells;

Note:Reagents should be adjusted proportionally according to the sample volume; set aside a pre-sorting sample to measure 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, gentle pipetting 2–3 times can be used to mix thoroughly.

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

Shake for 5–30 s to mix the Streptavidin beads thoroughly;

Mix for 5–30 s

6

Add 15 μL of Streptavidin beads to the sample;

15 μL / 100 μL

7

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

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

During this step, the magnetic beads may settle at the bottom; you can gently flick the tube to remix them around the 2.5-minute mark;

Incubate at room temperature for 5 minutes

8

Add MagSep Separation Buffer to the sample and immediately place it on the magnetic stand to prevent the beads from settling heavily at the bottom;

Bring the total volume up to 2.5 mL (for a 5 mL flow cytometry tube)

Add the total volume to 7.5 mL (using a 15 mL centrifuge tube).

9

Place the sample on the magnetic stand so that the magnetic beads adhere to the tube wall.

Adsorb using a single-well magnetic stand (3 min).

Adsorb using a multi-well magnetic stand (5 min).

10

Tilt the magnetic stand and pour the sample into a new collection tube to collect the sample.

Note: The sorted cells can be centrifuged at 400g for 7 minutes for subsequent culture and analysis.

Cell sorting successful.

Note:1. 0.1-1 × 108cells (0.1-1 mL) are recommended to use a 5 mL single-well magnetic separator (Starter EasyIso Separator) with a total adsorption volume of 2.5 mL;

2-5 × 108cells (2-5 mL) are recommended to use a 15 mL multi-well magnetic separator (Starter EasyEights EasyIso Separator) with a total adsorption volume of 7.5 mL;

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);


Picture

Validation Data

CD8 T cells were enriched from mouse splenocytes using a CD8 T‑cell column‑free isolation kit. Cells were stained with CD8‑Alexa Fluor® 647 (SDT Cat: S0B5895) and CD4‑Alexa Fluor® 488 (SDT Cat: S0B8030) fluorescent antibodies, and analysis was performed by gating on live cells (Live+), with the proportion of CD8 T cells in the sorted fraction being assessed. Flow cytometry results indicated that the purity of CD8 T cells after this sorting was 97.9%.