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Mouse T Cell Isolation Kit (Column-Based)

Mouse T Cell Isolation Kit (Column-Based)

Catalog Number: S0K1008 Reactivity: Mouse Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Mouse T Cell Biotin-Antibody Cocktail

0.5mL 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 T cells from mouse spleens. In this negative selection process, non-T cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin‑nanomagnetic beads. As a result, the unwanted cells become bound to the magnetic beads via the antibody‑antigen complex, while the target 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 T cells elute by gravity into the collection tube. The resulting supernatant represents the 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 T lymphocytes are the most central effector cell population in the murine adaptive immune system, serving as a key force in defending the body against pathogen invasion, eliminating abnormal cells, and maintaining immune homeostasis. The CD3 molecule is the most characteristic transmembrane glycoprotein complex on the surface of mouse T cells, composed of four polypeptide chains—γ, δ, ε, and ζ—joined by noncovalent bonds. Together with the T-cell receptor (TCR), they form the TCR–CD3 complex. Although the CD3 molecule does not directly recognize antigens, its primary function is to transmit the activation signals generated by TCR–antigen interactions into the cell, thereby initiating T-cell activation, proliferation, and differentiation. It is an essential prerequisite for T-cell maturation, activation, and functional execution; consequently, the CD3 molecule is often used as a specific surface marker for identifying T cells. In scientific research, obtaining highly pure, high‑activity mouse T cells is a prerequisite for conducting T‑cell‑related studies. This negative selection kit employs the principle of negative sorting: biotin‑conjugated antibodies specifically label non‑CD3⁺ T cells (such as B cells, macrophages, and granulocytes), which are then bound and removed using streptavidin‑coated nanomagnetic beads. Throughout the process, there is no binding to T‑cell surface antigens, allowing for maximum preservation of their native morphology, integrity of surface markers, and functional activity, thus providing reliable cellular material for subsequent experiments.

Protocol

Note: This kit does not include separation buffer; please 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 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 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 it evenly;

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 wellStreptavidin Beads, add 5 μLStreptavidin Beadsinto the sample;

5 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

Place theL Separation Columnin the column separation magnet, then 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 hold 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);


Picture

Validation Data

T cells were enriched from mouse splenocytes using the Mouse T-cell column-based sorting kit. Cells were stained with CD19-Alexa Fluor® 647 (SDT Cat: S0B5445) and CD3-Alexa Fluor® 488 (SDT Cat: S0B8364) fluorescent antibodies, and analysis was performed by gating on live cells (Live+), allowing for the determination of the proportion of T cells in the sorted fraction. Flow cytometry results indicated that the purity of T cells after this sorting step was 98.2%.