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Mouse T Cell Isolation Kit (Column-Free)

Mouse T Cell Isolation Kit (Column-Free)

Catalog Number: S0K3006 Reactivity: Mouse Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Mouse T Cell Biotin-Antibody Cocktail

0. 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

In this negative selection process, non- is labeled with a biotin-conjugated antibody.TCells are then incubated with streptavidin‑coated nanomagnetic beads; non‑target cells are labeled via antibody‑bead complexes, while the target…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 unlabeled cells…TThe cells remain in the supernatant. This supernatant is, therefore, the fraction obtained after removing the non-target cells.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 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—linked by non-covalent 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 magnetic nanoparticles. Throughout the process, there is no binding to T‑cell surface antigens, thus maximizing the preservation of their native morphology, the integrity of surface markers, and functional activity, providing reliable cellular material for subsequent experiments.

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

Take the mouse spleen, grind it on a 40 μm sterile filter to prepare a single-cell suspension of splenocytes; proceed with cell counting;

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

Sample Preparation

2

Resuspend 1×10 in 100μL MagSep Separation Buffer7cells;

Note:Reagents should be adjusted proportionally according to the sample volume; set aside a pre-sorting sample to determine the initial Mouse T ratio;

1×107cells/100 μL

3

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

Note:For 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 5 μL of Streptavidin beads to the sample;

5 μL/100 μL

7

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

Note:In this step, you can use a pipette to gently blow the mixture 2–3 times for mixing;

During this step, the magnetic beads may settle at the bottom; you can lightly tap 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 magnetic beads from settling heavily at the bottom;

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

Add the total volume to 7.5 mL (in 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 it;

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

Cell sorting successful

Note: 1.For 0.1–1 × 10^8 cells (0.1–1 mL), it is recommended to use a 5 mL single-well magnetic separator (Starter EasyIso Separator) with a total adsorption volume of 2.5 mL;

For 2–5 × 10^8 cells (2–5 mL), it is 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 live cell rate in the sample 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‑column‑free sorting kit. Cells were stained with CD19‑Alexa Fluor® 647 (SDT Cat: S0B5445) and CD3‑Alexa Fluor® 488 (SDT Cat: S0B8364), 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 cytometric analysis revealed that the purity of the sorted T cells was 98.4%.