CS-0051

Mouse CD4+CD25- Conventional T Cell Sorting Kit

Column-Free Negative Mouse

The Cell Sorts CS-0051 Mouse CD4+CD25- Conventional T Cell Sorting Kit uses column-free negative selection to isolate untouched CD4+CD25- conventional T cells (Tconv) from mouse spleen with up to 98% purity in 18–20 min. No RBC lysis is required — the optimized protocol saves time and preserves viability. Mouse CD4 Tconv Cell Isolation Cocktail (100 μL/mL) labels non-target cells at RT for 10 min, followed by Streptavidin Nanobeads (100 μL/mL) for 5 min. Place on a magnetic separator for 3–5 min, then simply pour off purified Tconv cells.

No columns, no centrifugation, no bead or antibody contact with target cells. This kit is colitis model optimized — specifically designed for mouse colitis model construction and IBD research. Isolated CD4+CD25- conventional T cells are immediately ready for Th subset analysis, effector T cell studies, autoimmunity models without Treg contamination, flow cytometry, scRNA-seq, and adoptive cell therapy.

Sorting Purity

Up to 98%

Sorting Time

18–20 min

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Product Features of the Mouse CD4+CD25- Conventional T Cell Sorting Kit

The Mouse CD4+CD25- Conventional T Cell Sorting Kit delivers industry-leading performance for rapid CD4+CD25- Conventional T Cell isolation from PBMCs via column-free negative selection.

Column-free separation — No column needed, streamlined workflow; simply pour target cells into a new tube

No RBC lysis required — Optimized protocol eliminates red blood cell lysis step, saving time and preserving cell viability

Zero contact with target cells — Antibodies and beads bind non-CD4 Tconv cells only, preserving CD4+CD25- T cells in their native, unactivated state

Purity up to 98% — High-efficiency negative selection for ultra-pure CD4 Tconv cell populations

18-20 min fast sorting — Rapid one-step protocol from mouse spleen to purified CD4 Tconv cells

Colitis model optimized — Specifically designed for mouse colitis model construction and IBD research

The Mouse CD4+CD25- Conventional T Cell Sorting Kit uses column-free negative selection to isolate untouched CD4+CD25- Conventional T Cells from human PBMCs. To achieve these results, follow this simple 4-step protocol that takes just 18–20 min from start to finish. No centrifugation and no lengthy incubations. Furthermore, this streamlined protocol ensures consistent results across experiments.

How the Mouse CD4+CD25- Conventional T Cell Sorting Kit Works

Simple 4-step protocol for rapid cell isolation

Step 1: Sample Preparation

Harvest mouse spleen, grind through a 70 μm sterile nylon mesh filter. Count cells and resuspend in FACS Buffer at 1×10⁸ cells/mL (e.g., 1×10⁷ cells in 100 μL). No RBC lysis required.

Step 2: Antibody Labeling

Add Mouse CD4 Tconv Cell Isolation Cocktail (100 μL/mL), mix gently by pipetting 2–3 times, incubate RT 10 min. Labels non-CD4 Tconv cells; CD4+CD25- conventional T cells untouched.

Step 3: Magnetic Bead Binding

Vortex Streptavidin Nanobeads 5–30 s, add 100 μL per mL of cell suspension, mix gently, incubate RT 5 min to bind labeled non-target cells.

Step 4: Column-Free Separation & Collection

Dilute with FACS Buffer to 2.5 mL (5 mL tube) or 7.5 mL (15 mL tube). Place on magnetic separator 3–5 min, then pour off untouched purified CD4+CD25- Tconv cells into a fresh collection tube.

Applications for the Mouse CD4+CD25- Conventional T Cell Sorting Kit

This Mouse CD4+CD25- Conventional T Cell Sorting Kit supports a wide range of downstream applications for CD4+CD25- Conventional T Cell isolation and column-free negative selection workflows from mouse splenocytes or lymph node cells.

Colitis Model Construction — Adoptive transfer of CD4+CD25⁻ T cells into immunodeficient mice to induce IBD models Autoimmune Disease Research — Study the pathogenic role of effector T cells in autoimmune conditions Treg Function Studies — Use as a control population alongside CD4+CD25+ Treg cells in suppression assays Immunology Research — CD4+ effector T cell activation, proliferation, and cytokine production studies Flow Cytometry — High-purity CD4 Tconv populations ready for CD4/CD25/Foxp3 staining and analysis Cell Culture — Unactivated, bead-free CD4 Tconv cells ideal for in vitro expansion and functional assays Drug Discovery — T cell-targeted drug screening in colitis and autoimmune mouse models

In addition, the Mouse CD4+CD25- Conventional T Cell Sorting Kit is compatible with a wide range of downstream applications, making it a versatile tool for your immunology research workflow.

Kit Components

Each Mouse CD4+CD25- Conventional T Cell Sorting Kit contains everything needed for high-purity column-free negative selection from human PBMCs.

Biotin-conjugated antibody cocktail
Streptavidin nanobeads
User manual

Storage & Shelf Life

Storage

Store at 2-8°C protected from light

Shelf Life

12 months

Mouse CD4+CD25- Conventional T Cell Sorting Kit — Frequently Asked Questions

Everything you need to know about the CS-0051 Mouse CD4+CD25- Conventional T Cell Sorting Kit

Click each question to expand the answer

The CS-0051 Mouse CD4+CD25- Conventional T Cell Sorting Kit from Cell Sorts uses column-free negative selection. Non-target cells (CD8+ T cells, B cells, NK cells, monocytes, granulocytes, erythrocytes, CD25+ regulatory T cells) are labeled with a biotin-conjugated antibody cocktail and removed by magnetic separation, while untouched CD4+CD25- conventional T cells remain in the supernatant. This ensures no beads or antibodies ever contact the target cells, preserving their native functional state.

Purity of isolated CD4+CD25- conventional T cells typically reaches up to 98%, as validated by flow cytometry using CD4-FITC (Clone GK1.5), CD25-PE (Clone PC61), CD3-APC, FoxP3 staining. The optimized negative selection protocol efficiently depletes all non-target cell populations while maximizing recovery of viable, functional CD4+CD25- conventional T cells from mouse spleen. No RBC lysis is required, preserving cell viability.

The CS-0051 kit is optimized for mouse spleen and lymph node cell suspensions. Prepare a single-cell suspension by gentle mechanical disruption through a 70 μm sterile nylon mesh filter. No red blood cell lysis is required — the optimized protocol eliminates the ACK lysis step, saving time and preserving cell viability. The kit is compatible with C57BL/6, BALB/c and other common mouse strains used in immunology and IBD research.

Step 1: Sample Preparation. Harvest mouse spleen, grind through a 70 μm sterile nylon mesh filter to prepare a single-cell suspension. Count cells and resuspend in FACS Buffer at 1×10⁸ cells/mL (e.g., 1×10⁷ cells in 100 μL). No RBC lysis required.
Step 2: Antibody Labeling. Add Mouse CD4 Tconv Cell Isolation Cocktail (100 μL/mL), mix gently by pipetting 2–3 times, incubate at room temperature for 10 min to label non-CD4 Tconv cells.
Step 3: Magnetic Bead Binding. Vortex Streptavidin Nanobeads for 5–30 s, add 100 μL per mL of cell suspension, mix gently, incubate at room temperature for 5 min.
Step 4: Column-Free Separation & Collection. Dilute with FACS Buffer to 2.5 mL (5 mL tube) or 7.5 mL (15 mL tube). Place on magnetic separator for 3–5 min, then pour off the untouched, purified CD4+CD25− Tconv cells into a fresh collection tube.
Total time: 18–20 min.

Isolated CD4+CD25- conventional T cells are immediately ready for: conventional T cell research, Th subset analysis, effector T cell studies, autoimmunity models without Treg contamination, flow cytometry analysis, cell culture and expansion, CRISPR gene editing, single-cell RNA sequencing (scRNA-seq), and adoptive cell therapy studies. Zero bead or antibody interference means no additional washing steps are required.

Antibodies and streptavidin nanobeads bind exclusively to non-target cells—they never touch CD4+CD25- conventional T cells. This untouched approach ensures full preservation of surface receptors, native functional states, and physiological relevance for downstream functional assays. Cell Sorts column-free technology eliminates column-induced mechanical stress, further enhancing cell viability.

The antibody cocktail targets CD8a, CD19, CD11b, CD49b, Ly-6G, Ter-119, CD25 on non-target cells—effectively labeling CD8+ T cells, B cells, NK cells, monocytes, granulocytes, erythrocytes, CD25+ regulatory T cells for depletion while leaving CD4+CD25- conventional T cells completely untouched. This comprehensive marker panel ensures high purity and recovery of the target cell population.

We recommend flow cytometry using CD4-FITC (Clone GK1.5), CD25-PE (Clone PC61), CD3-APC, FoxP3 antibodies. Typical results show up to 98% CD4+CD25- conventional T cells in the collected fraction. Viability can be assessed using Trypan blue exclusion or 7-AAD staining. For best results, gate on lymphocytes by forward and side scatter, then analyze marker expression within the gated population.

Absolutely. Since isolated CD4+CD25- conventional T cells are completely bead-free and antibody-free, they are immediately compatible with single-cell RNA sequencing (scRNA-seq) and other single-cell technologies without additional washing steps. Cell Sorts protocols are validated for 10x Genomics and other major scRNA-seq platforms.

The CS-0051 uses column-free negative selection (18–20 min) with no RBC lysis required, ideal for speed and convenience. Column-based kits (25–35 min) offer higher consistency for larger sample sizes. Both yield untouched, bead-free CD4+CD25- conventional T cells. The CS-0051 is also colitis model optimized — specifically designed for mouse colitis model construction and IBD research. Choose CS-0051 for rapid processing and colitis/IBD studies; choose column-based for larger-scale isolation.