Naïve CD4 T cells were enriched from mouse splenocytes using a column-free Naïve CD4 T cell isolation kit. Cells were stained with CD4-Pacific Blue, CD44-FITC, and CD62L-Alexa Fluor® 647 fluorescent antibodies, and analysis was performed by gating on live cells (Live+). The proportion of Naïve CD4 T cells (CD4+CD44-CD62L+) in the sorted fraction was determined; flow cytometry results indicated that the purity of the Naïve CD4 T cells after this sorting was 95.5%.
Mouse Naïve CD4 T Cell Isolation Kit (Column-Free)
Mouse Naïve CD4 T Cell Isolation Kit (Column-Free)
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Product Details
Product Details
Product Specification
| Format |
1 mL Mouse Naïve CD4 T Cell Biotin-Antibody Cocktail 1mL 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-target cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-nanomagnetic beads. The non-target cells are marked by the antibody‑bead complex, while the targetNaïve CD4 Tcells remain unlabeled. Subsequently, the cell suspension is 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, whereas the unlabeledNaïve CD4 Tcells remain in the supernatant. This supernatant constitutes theNaïve CD4 Tcell-enriched fraction after removal of the non-target cells.Naïve CD4 Tcell 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 naïve CD4 T cells refer to mature CD4 T cells that have never encountered a specific antigen and remain in a quiescent state. They serve as the common precursor for all effector CD4 T cell subsets (such as Th1/Th2/Th17/Treg) and are a model system for studying the mechanisms of T cell activation, differentiation, and function. Using a column-free Mouse Naïve CD4 T cell isolation kit, antibody-naïve, initial CD4 T cells can be rapidly and efficiently purified. This method removes only non-target cells during the sorting process without labeling or activating the target naïve CD4 T cells, thereby preserving their native phenotype and functional integrity, making them suitable for subsequent cell culture and functional assays.
Components
Protocol
Note: This kit does not include sorting buffer; please prepare it yourself. The corresponding Stater catalog number isS0D3018
Steps |
Operating Instructions |
Dosage and Time |
1 |
Collect the mouse spleen, grind it on a 40 μm sterile filter to prepare a single-cell suspension of splenic cells; after red blood cell lysis, proceed with cell counting. Note: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 1×10 in 100 μL of MagSep Separation Buffer7cells; Note:Adjust reagents proportionally according to sample volume; retain a pre-sorting sample to measure the Naïve CD4 ratio. |
1×107cells/100 μL |
3 |
Add 10 μL of Mouse Naïve CD4 T cell Biotin Antibody Cocktail to the sample and gently mix. Note:For this step, gently pipette up and down 2–3 times to mix thoroughly; |
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 |
Shake for 5–30 seconds to mix the Streptavidin beads evenly; |
Mix for 5–30 seconds |
6 |
Add 10 μL of Streptavidin beads to the sample; |
10 μL/100 μL |
7 |
Gently mix the magnetic beads and cells, and incubate at room temperature for 5 minutes; Note:For this step, you can use a pipette to gently blow the mixture 2–3 times to mix it evenly; 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 beads from settling heavily at the bottom; |
Add the total volume to 2.5 mL (in a 5 mL flow cytometry tube) |
9 |
Place the sample on a single-well magnetic stand so that the magnetic beads adhere to the tube wall; |
Hold the sample on the single-well magnetic stand for 10 minutes |
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:0.1-1 × 108cells (0.1–1 mL) are recommended to use a 5 mL single-well magnetic separator (Starter EasyIso Separator) to adsorb a total volume of 2.5 mL; | ||
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Validation Data
