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Cat. No.: HY-K0308

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Mouse CD4⁺ Cell Positive Selection Kit

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MCE Mouse CD4⁺ Cells Positive Selection Kit is designed for the isolation of CD4⁺ cells from single cell suspensions of mouse spleen cells, lymph nodes or other tissues.

Storage :
4°C，2 years

Do not freeze the magnetic beads

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Use of MCE Products

Fluorescent Dye

Strong Fluorescence Signal / Multi-Disciplinary Applications / High Fluorescence Specificity

Description & Advantages

MCE Mouse CD4⁺ Cells Positive Selection Kit is designed for the isolation of CD4⁺ cells from single cell suspensions of mouse spleen cells, lymph nodes or other tissues. Its principle is that CD4 Capture Antibody can label CD4⁺ cells, these target cells are captured by Releasable Magnetic Beads, and finally, the Magnetic Beads Release Buffer dissociates the magnetic beads from the cell surface. This process effectively facilitates the sorting of mouse CD4⁺ cells that are not labelled with magnetic beads.

MCE Mouse CD4⁺ Cells Positive Selection Kit is user-friendly and portable, with components that are non-toxic to cells. It enables the isolation of high-purity cells, and the resulting cells can be utilized in a variety of downstream applications. These include flow cytometry, cell culture, Western blot analysis, immunoprecipitation (IP), reporter gene detection, and DNA/RNA extraction.

Features of MCE Mouse CD4⁺ Cells Positive Selection Kit

• Separation Columns-free: Eliminates the need for separation columns by using a magnetic separator for swift cell separation;

• High Purity: Can yield sorted cells with a purity exceeding 95%;

• Broad Applicability: Can be used directly for the capture of target cells, and the final cells obtained are free of magnetic bead labeling.

Protocol

Recommended Buffer (Not Provided)

Buffer	Composition
Isolation Buffer	PBS, 0.5% BSA, 2 mM EDTA, pH 7.0-7.4

Note: a. BSA may be replaced with human serum albumin (HSA) or 2% fetal bovine serum (FBS);
b. It is recommended to prepare the Isolation Buffer with water for injection, sterilize it through a 0.22 μm filter, and store it at 4°C.

Example: Isolation of mouse CD4⁺ cells from mouse spleen, lymph nodes, or other tissues

1. Prepare a single-cell suspension from mouse spleen, lymph nodes, or other tissues: dissociate the tissue through a 70 μm cell strainer and rinse the strainer with pre-cooled PBS. Transfer the cell suspension to a 50 mL centrifuge tube, centrifuge at 500 × g for 5 min, and discard the supernatant.

2. Add 5 mL ACK Red Blood Cell Lysis Buffer and lyse at room temperature for 5 min. Add 20 mL PBS to resuspend the cells, centrifuge at 500 × g for 5 min, and discard the supernatant.

3. Resuspend the cells in PBS, filter through a 70 μm cell strainer, and count the cells. Centrifuge at 500 × g for 5 min and discard the supernatant.

4. Resuspend the cells in Isolation Buffer and adjust the cell density to 1 × 10⁸ cells/mL.

5. Transfer 100 μL of cell suspension (1 × 10⁷ cells) to the bottom of a sterile centrifuge tube. Add 2 μL CD4 Capture Antibody, mix thoroughly, and incubate at 4°C for 10 min.

Note: Scale the amount of capture antibody proportionally for larger cell numbers. For fewer than 1 × 10⁷ cells, adjust the cell suspension volume to 100 μL.

6. Preparation of Releasable Magnetic Beads: thoroughly resuspend the beads and transfer 20 μL to a 1.5 mL microcentrifuge tube. Add 1 mL Isolation Buffer, mix, centrifuge at 10,000 × g for 1 min, and discard the supernatant. Repeat the wash 1-2 times and finally resuspend the beads in 20 μL Isolation Buffer.

7. After antibody incubation, add 20 μL of pre-washed Releasable Magnetic Beads, mix thoroughly, and incubate at 4°C for 10 min.

8. Add 2.5 mL Isolation Buffer and mix gently. Place the tube on a magnetic separation rack and allow it to stand for 5 min.

9. While the tube remains on the magnetic rack, carefully discard the supernatant. Remove the tube from the rack, add 2.5 mL Isolation Buffer, and thoroughly resuspend the bead-cell complexes.

10. Place the tube on the magnetic rack again for 5 min and discard the supernatant. Repeat Steps 9-10 for a total of two washes.

11. Remove the tube from the magnetic rack, add 400 μL Magnetic Beads Release Buffer, gently resuspend the bead-cell complexes, and incubate at room temperature for 10 min. Mix gently every 2-3 min during incubation.

12. Add 2.5 mL Isolation Buffer, mix gently, and place the tube on the magnetic rack for 5 min.

13. While keeping the tube on the magnetic rack, carefully collect the supernatant into a new sterile centrifuge tube. The supernatant contains bead-free mouse CD4⁺ cells. Centrifuge at 300 × g for 5 min, discard the supernatant, and collect the cells.

14. Wash the cells as required and resuspend them in an appropriate buffer or culture medium. The isolated cells may be used for downstream experiments.