Naïve CD4 T cells were enriched from mouse splenocytes using a column-based sorting kit. Cells were stained with the fluorescent antibodies CD4-Pacific Blue (SDT Cat: S0B5831), CD44-FITC (SDT Cat: S0B5452), and CD62L-Alexa Fluor® 647 (SDT Cat: S0B1699). Gating was performed on live cells (Live+), and the proportion of Naïve CD4 T cells (CD4+CD44-CD62L+) in the sorted fraction was determined. Flow cytometry analysis revealed that the purity of the sorted Naïve CD4 T cells was 93.7%.
Mouse Naïve CD4 T Cell Isolation Kit (Column-Based)
Mouse Naïve CD4 T Cell Isolation Kit (Column-Based)
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Product Details
Product Details
Product Specification
| Format |
1 mL Mouse Naïve CD4 T Cell Biotin-Antibody Cocktail 1mL Streptavidin Beads 1mL Mouse CD44 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 column-based negative selection method to isolate naïve CD4 T cells from mouse spleens. In this negative selection process, non‑naïve CD4 T cells are labeled with biotin‑conjugated antibodies, and the cells are then incubated with streptavidin‑nanomagnetic beads. As a result, the non‑target cells become bound to the magnetic beads via the antibody complexes, while the target naïve CD4 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 naïve CD4 T cells elute by gravity into the collection tube. The resulting supernatant represents the enriched fraction of naïve CD4 T cells after removal of the non‑target cells. |
| Applications in cell sorting | Naïve CD4 T cells in their unmanipulated, native state are sorted from a single-cell suspension of fresh mouse spleen and can be used for a variety of downstream functional assays. |
| 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 naive CD4⁺ T cells (initial CD4-positive T lymphocytes) are mature CD4⁺ T cells that have not yet encountered specific antigens and have not undergone activation, proliferation, or differentiation. By flow cytometry, their canonical phenotype is defined as CD4⁺CD44⁻CD62L⁺. CD62L (L-selectin) is a key receptor for lymph node homing, highly expressed on quiescent naive T cells; upon cell activation or exposure to mechanical or thermal stress, it is rapidly cleaved and shed by the ADAM17 metalloprotease. CD44 is an adhesion molecule characteristic of activated and memory T cells, and is barely expressed on resting naive CD4⁺ T cells. Based on these features, naive T cells (CD44⁻CD62L⁺), central memory T cells (CD44⁺CD62L⁺), and effector memory T cells (CD44⁺CD62L⁻) can be precisely distinguished. Cell purity is a critical prerequisite for functional assays involving naive CD4⁺ T cells. This kit employs immunomagnetic bead-based negative selection: biotinylated antibodies specifically label non-target cells, including B cells, CD8⁺ T cells, myeloid cells, and activated/memory T cells, which are then selectively removed using streptavidin-conjugated magnetic beads. Throughout the process, the target naive CD4⁺ T cells remain unlabeled and untouched, thereby maximizing preservation of their quiescent phenotype, viability, and differentiation potential. This approach efficiently yields high-purity, highly active, unactivated "untouched" naive CD4⁺ T cells, suitable for various experimental applications such as in vitro polarization, cell activation assays, and studies of immune mechanisms.
Protocol
NoteThis kit does not include sorting buffer; please provide it separately. The corresponding Stater product number is:S0D3018.
withL Separation ColumnFor example
Steps |
Operating Instructions |
Dosage and timing |
|
Cell processing and labeling |
1 |
The spleen was harvested from mice, passed through a 40-μm sterile filter to prepare a single-cell suspension, and subjected to red blood cell lysis using Red Blood Cell Lysis Buffer (without fixative, 1X) (Cat: S0D9002) prior to cell counting. Note:Throughout the grinding, filtration, and washing steps, use pre-cooled reagents and handle samples gently; avoid vigorous vortexing. It is recommended to count cells after staining with AO/PI. For splenic cells, centrifuge at 400 g for 7 minutes. |
Prepare the sample |
2 |
Using 100 μL of pre-cooledMagSep Separation BufferResuspend to 1 × 10⁷cell; Note:Reagents are adjusted proportionally based on the sample volume; retain a pre-sorting sample for testing the naïve CD4 percentage. |
1 × 10⁷cells/100 μL |
|
3 |
Add 10 μL of the Mouse Naive CD4 T cell Biotin Antibody Cocktail to the sample and gently mix. Note:In this step, the mixture can be gently mixed by pipetting up and down 2–3 times. |
10 μL/100 μL |
|
4 |
Incubate the antibodies and cells in the cocktail at 2–8°C for 10 minutes. |
Incubate at 2–8°C for 10 min. |
|
5 |
Add 10 times the cell volume of pre-cooledMagSep Separation BufferWash the cells, centrifuge at 400g for 7 minutes, discard the supernatant, and resuspend to the original volume. |
Centrifuge at 400g for 7 minutes. |
|
6 |
Mix the Streptavidin Beads thoroughly, then add 10 μL of Streptavidin Beads to the sample. |
10 μL/100 μL |
|
7 |
Mix the Mouse CD44 Nanobeads well, then add 10 μL of Mouse CD44 Nanobeads to the sample; |
10 μL/100 μL |
|
8 |
Gently mix the magnetic beads and cells, and incubate at 2–8°C for 5 minutes; |
Incubate at 2–8°C for 5 minutes |
|
Cell sorting |
9 |
In the column-based magnetic separator, install the L Separation Column and add 3 mLMagSep Separation Bufferto 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 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 to collect the target cells; Note:This step elutes and sorts the unlabeled target cells, increasing the yield. |
Collect the target cells |
|
|
Notes:1. Pay attention to the maximum capacity of the separation column: the S Separation Column can handle a maximum of 1×10⁷ labeled cells;the L Separation Column can handle a maximum of 1×10⁸ labeled cells.; 2. Throughout the entire process, carefully control the temperature. Keep the sorting buffer pre-cooled at all times, and proceed with subsequent experiments immediately after sorting. For flow cytometry, perform staining in the dark at 2–8°C to avoid prolonged exposure at room temperature. | |||
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Validation Data
