CD4 T cells were enriched from mouse splenocytes using a CD4 T‑cell column‑free isolation kit. Cells were stained with CD8‑Alexa Fluor® 647 (SDT Cat: S0B5895) and CD4‑Alexa Fluor® 488 (SDT Cat: S0B8030) fluorescent antibodies, and analysis was performed by gating on live cells (Live+), with the proportion of CD4 T cells in the sorted fraction being assessed. Flow cytometry results indicated that the purity of CD4 T cells after this sorting was 95.8%.
Mouse CD4 T Cell Isolation Kit (Column-Free)
Mouse CD4 T Cell Isolation Kit (Column-Free)
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Product Details
Product Details
Product Specification
| Format |
1 mL Mouse CD4 T Cell Biotin-Antibody Cocktail 0.75 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.CD4 TCells are then incubated with streptavidin‑coated nanomagnetic beads; non‑target cells are labeled via antibody complexes bound to the beads, while the target…CD4 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 unlabeledCD4 TThe cells remain in the supernatant. This supernatant is, therefore, the solution from which non-target cells have been removed.CD4 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 CD4 T lymphocytes are a core functional cell subset of the murine adaptive immune system. They originate from hematopoietic stem cells in the bone marrow, complete their development and maturation in the thymus, and then migrate to secondary lymphoid organs such as the spleen and lymph nodes. These cells specifically express the CD4 molecule on their surface and can recognize antigen peptides presented by antigen‑presenting cells (APCs) in complex with MHC class II molecules via the T‑cell receptor (TCR), thereby becoming activated and exerting immunoregulatory functions; they are often referred to as the "command cells" of the immune system. In scientific research, obtaining highly purified, high‑activity mouse CD4 T cells is essential for conducting related studies. This negative selection kit employs a negative selection strategy: biotin‑conjugated antibodies label non‑CD4⁺ T cells, which are then bound by streptavidin‑coated magnetic nanoparticles and removed, keeping CD4 T cells untouched throughout the process. This approach maximizes the preservation of their native morphology 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 |
Collect the mouse spleen, grind it on a 40 μm sterile filter to prepare a single-cell suspension of splenic cells; perform 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; |
Prepare the sample |
2 |
Resuspend 1×10 in 100μL MagSep Separation Buffer7cells; Note:Adjust reagents proportionally according to the sample volume; set aside a pre-sorting sample to measure the initial CD4 ratio; |
1×107cells/100 μL |
3 |
Add 10 μL Mouse CD4 T cell Biotin Antibody Cocktail to the sample and gently mix. Note:In this step, gentle pipetting 2–3 times can be used to mix evenly. |
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 7.5 μL of Streptavidin beads to the sample; |
7.5 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 gently tap the tube to remix them after about 2.5 minutes. |
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 (for a 5 mL flow cytometry tube). Add the total volume to 7.5 mL (using 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 the sample; 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); | ||
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Validation Data
