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 assessed. Flow cytometry analysis revealed that the purity of the Naïve CD4 T cells after sorting 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. |
| 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 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 of all effector CD4 T cell subsets (such as Th1, Th2, Th17, and Treg) and are a model system for studying the mechanisms of T cell activation, differentiation, and function. Using the column-based 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 isolation 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.
Protocol
Note: This kit does not include separation buffer; please prepare it yourself. The corresponding Stater catalog number isS0D3018
TakingL Separation Columnas an example
Steps |
Operating Instructions |
Dosage and Time |
|
|
Cell Processing and Labeling |
1 |
Take the mouse spleen, grind it on a 40 μm sterile filter to prepare a single-cell suspension of the spleen, perform red blood cell lysis, and then count the cells. 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 1×10^7 cells in 100 μL of MagSep Separation Buffer.7cells; Note:Adjust reagents proportionally according to the sample volume; set aside a pre-sorting sample to determine the naïve CD4 ratio. |
1×107cells/100 μL |
|
3 |
Add 10 μL of Mouse Naive 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; |
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 |
Add MagSep Separation Buffer equal to ten times the cell volume to wash the cells; after centrifuging at 400g for 7 minutes, discard the supernatant and resuspend the cells to their original volume; |
Centrifuge at 400g for 7 minutes |
|
6 |
Mix wellStreptavidin Beads, add 10 μLStreptavidin Beadsto the sample; |
10 μL/100 μL |
|
7 |
Mix wellMouse CD44 Nanobeads, add 10 μLMouse CD44 Nanobeadsinto the sample; |
10 μL/100 μL |
|
8 |
Gently mix the magnetic beads and cells, and incubate at room temperature for 5 minutes; |
Incubate at room temperature for 5 minutes |
|
Cell sorting |
9 |
In the column separation magnet, installL Separation Column, add 3 mL of MagSep Separation Buffer to 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 500 μL; if insufficient, make up the volume with MagSep Separation Buffer; the cells that drip down under gravity are the target cells; |
Collect the target cells |
|
11 |
Add 3 mL of MagSep Separation Buffer and collect the target cells; Note:This step elutes the unlabeled target cells from the separation column, increasing the yield. |
Collect the target cells |
|
|
Notes:1. Pay attention to the maximum capacity of the separation column,The S Separation Column can label a maximum of1×107;The maximum number of labeled cells for the L Separation Column is1×108; 2. If the sample viability 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
