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Mouse CD4+ CD25+ Regulatory T Cell Isolation Kit ( Column-Based)

Mouse CD4+ CD25+ Regulatory T Cell Isolation Kit ( Column-Based)

Catalog Number: S0K1022 Reactivity: Mouse Conjugation: Brand: Starter
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$1,335 USD
$1,335 USD
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Product Details

Product Specification


Format

1 mL Mouse CD4+ CD25+ Regulatory T Cell Biotin-Antibody Cocktail

1 mL Streptavidin NanoBeads

0.2 mL Mouse CD25-APC Antibody

1 mL Anti-APC Nanobeads
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 two-step immunomagnetic bead separation strategy to isolate CD4+ CD25+ regulatory T cells from mouse spleens. In the first step, CD4+ T cells are negatively enriched: non-CD4+ cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-nanoparticle magnetic beads. Non-target cells become labeled via antibody–bead complexes, while the target CD4+ T cells remain unlabeled. The cell suspension is then transferred to a separation column; under a magnetic field, the magnetically labeled non-target cells are retained in the column, whereas the unlabeled CD4+ T cells elute into the collection tube under gravity. This supernatant constitutes the CD4+ T cell-enriched fraction after removal of non-target cells. By first depleting a large number of contaminating cells, background interference in the subsequent CD25-positive selection is reduced. In the second step, CD25+ Tregs are positively selected: a CD25-APC specifically fluorescently labeled antibody is added to the enriched CD4+ T cell suspension, which binds specifically to CD25 molecules on the cell surface. Then, the matching anti-APC magnetic beads are added to label CD25+ cells via a bridging mechanism. The cells are again subjected to magnetic separation: CD4+ CD25+ cells are captured by the magnetic beads and retained in the magnetic field, while CD4+ CD25− conventional T cells are discarded with the supernatant. The eluted magnetic bead-bound fraction represents the target CD4+ CD25+ Treg cells.
Applications in cell sorting CD4⁺CD25⁺ Treg cells can be sorted from single-cell suspensions of fresh mouse spleens and used for a variety of downstream functional assays.
Reactivity Mouse
Magnetic Bead Size 20 nm
Separation Method Column-Based
Selection Strategy Positive Selection, Negative Selection
Stability & Storage

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

Background

Regulatory T cells (Tregs) are a key CD4⁺ T cell subset that maintain immune homeostasis and mediate peripheral immune tolerance. In 1995, Sakaguchi's team first demonstrated that the CD4⁺CD25⁺ population possesses potent immunosuppressive functions. Murine natural Tregs (nTregs) primarily develop in the thymus and are characterized by high expression of the IL-2 receptor α chain, CD25, with Foxp3 serving as their lineage-defining transcription factor. They suppress the excessive activation of effector T cells and antigen-presenting cells through mechanisms such as secreting inhibitory cytokines like TGF-β and IL-10, competitively depleting IL-2, and contact-dependent inhibition, thereby playing a central regulatory role in autoimmune diseases, transplant rejection, antitumor immunity, infectious immunity, and maternal-fetal tolerance.

CD25 is not an absolutely specific marker for Tregs; activated conventional CD4⁺ effector T cells (Tconv) also induce CD25 expression. Therefore, CD4⁺CD25⁺ represents a classic enrichment phenotype, while Foxp3 is the gold-standard marker for Treg identification. After sorting, it is recommended to confirm purity and identity by flow cytometry using combined staining for CD4, CD25, and Foxp3. Mouse spleen and lymph nodes are the primary sources for isolating CD4⁺CD25⁺ Tregs; this population typically accounts for 5%–12% of total CD4⁺ T cells in mice, with relatively low abundance, necessitating specialized immunomagnetic bead enrichment protocols to obtain highly active, high-purity Tregs suitable for in vitro suppression assays, expansion, transcriptomic studies, and adoptive cell transfer.

Protocol

Note:This kit does not include the sorting buffer; it must be provided separately. The corresponding Stater product number isS0D3018;

Note:It is recommended that the minimum initial number of sorted cells be 2 × 10⁷cells. A lower cell number may affect cell purity and yield. The amounts of antibodies and magnetic beads listed in the table below are calculated based on 2 × 10⁷cells;

Steps

Operating instructions

Dosage and time

Cell processing and labeling

1

Collect the mouse spleen, grind it on a 40 μm sterile filter to prepare a single-cell suspension of splenic cells, then perform red blood cell lysis using Red Blood Cell Lysis Buffer (without fixative, 1X) (Cat: S0D9002), followed by cell counting;

Note:Use pre-cooled reagents throughout the grinding, filtration, and washing processes, and perform all operations gently; avoid vigorous vortexing. 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 2 × 10⁷ live cells in 100 μL of pre-cooled MagSep Separation Buffer (Cat: S0D3018).Note: Adjust the volume of reagents proportionally according to the sample volume.

2 × 10⁷ cells / 100 μL

3Add 20 μL of Mouse CD4+ CD25+ Regulatory T Cell Biotin-Antibody Cocktail to the sample and mix gently.

Note:

For this step, gently pipette up and down 2–3 times to mix.

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

20 μL/100 μL

4

Incubate the antibodies in the cocktail with the cells at 2-8°C for 10 minutes.

Incubate at 2-8℃ for 10 min

5

Add pre-cooled MagSep Separation Buffer at 10 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

Add 4 μL of Mouse CD25-APC Antibody to the sample.

4 μL/100 μL

7

Mix the Streptavidin NanoBeads well, then add 20 μL of Streptavidin NanoBeads to the sample.

20 μL/100 μL

8

Gently mix the magnetic beads and cells, and incubate in the dark at 2-8℃ for 5 min.

Note:For this step, gently pipette up and down 2-3 times to mix.

Incubate in the dark at 2-8℃ for 5 min

Negative selection of cells

9

In the column separation magnet, install the L Separation Column and add 3 mL.MagSep Separation Buffer, rinse the separation column;

Rinse the separation column

10

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

Note:The cell volume should be at least 1 mL; if it is insufficient, useMagSep Separation Bufferto make up the volume; the cells that drip down under gravity are the target cells;

Collect the target cells

11

Add 3 mL ofMagSep Separation Bufferthree times, and collect the target cells;

Note:This step elutes the unlabeled target cells from the separation column, increasing the yield.

Collect the target cells

Positive cell selection

12

Take the eluate obtained in Step 11, centrifuge at 400g for 7 minutes, discard the supernatant, and resuspend the target cells in 100 μL ofMagSep Separation BufferResuspend the target cells

Note:For samples with fewer than 1×10⁸ cells before sorting,always resuspend in 100 μL

Resuspend in 100 μL

13

Mix the Anti-APC NanoBeads thoroughly, then add 20 μL of Anti-APC NanoBeads to the sample.

Note:Add Anti-APC NanoBeads based on the number of cells prior to sorting, at 10 μL per 10⁷ cells.cells

20 μL/2×10⁷cells

14

Gently mix the magnetic beads and cells, and incubate in the dark at 2–8°C for 15 minutes.

Note:In this step, the mixture can be gently mixed by pipetting up and down 2–3 times.

Incubate in the dark at 2–8°C for 15 minutes.

15

Add MagSep Separation Buffer, pre-cooled and at 10 times the cell volume, to wash the cells. After centrifuging at 400g for 7 minutes, discard the supernatant and resuspend the cells to the original volume.

Centrifuge at 400g for 7 minutes.

16

In the column selection magnet, install the S separation column and add 500 μL.MagSep Separation Buffer, rinse the separation column;

Rinse the separation column

17

Add the cells to the separation column and collect the flow-through in a collection tube;

Note:The cell volume should be at least 0.5 mL; if it is less, useMagSep Separation Bufferto make up the volume; the cells that drip down under gravity are the target cells;

Collect the flow-through

18

Add 500 μL ofMagSep Separation Bufferfour times; all the effluent constitutes the negative fraction; remove the separation column from the magnetic field and place it over a collection tube.

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19

Add 1 mL ofMagSep Separation Bufferinto the column, then quickly and firmly push with the accompanying plunger to elute the magnetically labeled positive cells;

Collect the target cells

20

To achieve higher purity, repeat steps 16–19; take the positive cells eluted in step 19 and perform a second column separation.

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Notes: 1. Pay attention to the maximum loading capacity of the separation column; the S Separation Column has a maximum labeled cell count of 1×10⁷.The L Separation Column has a maximum labeled cell count of 1×10⁸.;

2. Throughout the entire process, carefully control the temperature and avoid light exposure. Keep the separation buffer pre-cooled at all times and prevent it from being left at room temperature for extended periods.


Picture

Validation Data

CD4⁺CD25⁺ regulatory T cells were enriched from mouse splenocytes. Surface antigen staining was performed using the CD4-Alexa Fluor® 488 fluorescent antibody (SDT Cat: S0B8030), and intracellular antigen staining was performed using the FOXP3-Pacific Blue fluorescent antibody (SDT Cat: S0B8592). Analysis was conducted by gating on live cells (Live+), and the proportion of CD4⁺CD25⁺ regulatory T cells (CD4+CD25+FOXP3+) in the sorted fraction was determined. Flow cytometry results showed that after this sorting, the purity of CD4+ cells was 97.7%, and the purity of CD25+FOXP3+ cells was 88.1%.