Mouse Neutrophil Negative Selection Kit 

MCE Mouse Neutrophil Negative Selection Kit can isolate neutrophils from single-cell suspensions of mouse bone marrow or other tissue samples. The principle involves using biotin-labeled monoclonal antibodies to label non-target cells (non-CD11b+ Ly-6G+ cells), followed by the removal of these non-target cells through streptavidin-labeled magnetic beads, achieving the selective isolation of mouse neutrophils.

The kit is user-friendly and portable, with components that are non-toxic to cells. It enables high-purity cell separation and is suitable for isolating neutrophils from mouse bone marrow, peripheral blood, or spleen. The isolated cells can be utilized in a variety of downstream applications such as flow cytometry, cell culture, Western blot analysis, immunoprecipitation (IP), reporter gene detection, and DNA/RNA extraction.

Features of MCE Mouse Neutrophil Negative Selection Kit

1. Efficiency: Enables sorting of target cells within 30 min.

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

3. High Purity: The purity of the sorted cells can reach 95%.

4. High Activity: The sorted cells have high viability and good functionality, making them suitable for downstream experiments.

4℃, 2 years

Do not freeze the magnetic beads

1. During the use and storage of the Biotin-Antibody Mix, a void repeated freeze-thaw cycles, high-speed centrifugation, and other similar operations.

2. It is recommended to use low-adsorption consumables, such as pipette tips and centrifuge tubes to minimize loss of magnetic beads and antibodies due to adsorption.

3. During use and storage, magnetic beads should not be subjected to high-speed centrifugation, drying, or freezing. Avoid leaving magnetic beads in a magnetic field for extended periods, as this can cause bead aggregation, reducing their binding activity.

4. Before drawing magnetic beads from the storage tube, thoroughly mix them by gentle vortexing. During operation, handle gently and avoid creating air bubbles.

5. This pruduct should be used with a magnetic separator.

6. Perform all experimental procedures gently to avoid mechanical damage to the cells.

7. This product is for R&D use only, not for drug, household, or other uses.

8. For your safety and health, please wear a lab coat and disposable gloves to operate.

1. Red Blood Cell Lysis: Place the collected mouse bone marrow, spleen, or peripheral blood samples into a centrifuge tube and add red blood cell lysis buffer (MCE Cat. No.: HY-K3010) to lyse the red blood cells.

Note: a. It is recommended to adjust the volume and lysis time of the red blood cell lysis buffer based on the sample type. Mouse peripheral blood typically requires a longer lysis time.

b. A small amount of residual red blood cells will not affect subsequent sorting or cell purity.

c. Red blood cell lysis can be omitted for mouse bone marrow samples, but residual red blood cells may reduce neutrophil purity post-sorting (< 5%).

2. Prepare Single-Cell Suspension: After lysis, resuspend the cells in PBS and filter the cell suspension through a 70 μm cell strainer and count the cells. After counting, centrifuge at 500 × g for 5 min and discard the supernatant.

Note: The purpose of this step is to remove tissue and cell clumps from the cell suspension to prevent affecting the purity of subsequent cell sorting.

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

1. Beads Preparation: Resuspend the Streptavidin Magnetic Beads thoroughly and transfer 20 μL to a 1.5 mL EP tube. Add 1 mL of Isolation Buffer, mix and centrifuge at 10,000 × g for 1 min. Discard the supernatant and repeat wash the beads 1-2 times, resuspend the beads in 20 μL of Isolation Buffer.

Note: The final volume of Isolation Buffer used for the beads resuspension should be equal to the initial volume of beads that was aspirated.

2. Antibody Labeling: Transfer 100 μL of cell suspension (1 × 10⁷ cells) to the bottom of a flow tube. Then add 2 μL of Biotin-Antibody Mix, mix well and incubate at 4°C for 15 min.

Note: a. Transfer cell suspension directly to the bottom of the tube, avoiding adding along the wall of the tube.

b. If a larger quantity of cells requires sorting, the amount of Biotin-Antibody Mix can be increased proportionally.

3. Cell Washing: After the cell-antibody incubation, wash the cells once with Isolation Buffer, and add Isolation Buffer to 1.5 mL. Centrifuge at 500 × g for 5 min and discard the supernatant.

Note: If a larger quantity of cells requires sorting, it is recommended to use a 15 mL centrifuge tube and add Isolation Buffer to a final volume of 5-10 mL.

4. Magnetic Beads Labeling: After centrifugation, resuspend the cells in 100 μL of Isolation Buffer. Add 20 μL of pre-washed magnetic beads, mix well and incubate at 4°C for 10 min.

Note: If a larger quantity of cells requires sorting, the amount of magnetic beads can be increased proportionally. For sorting 5 × 10⁷ cells, add 10 μL of Biotin-Antibody Mix and 100 μL of beads to 500 μL of cell suspension. If sorting less than 1 × 10⁷ cells adjust the volume of the cell suspension to 100 μL and add 2 μL of Biotin-Antibody Mix along with 20 μL of beads.

5. Washing: After incubation, add 2.5 mL of Isolation Buffer to the flow tube and gently pipette to mix (avoid vigorous shaking or inverting).

6. Isolation: Place the flow tube containing cells on the magnetic separator for 5 min.

7. Collect Cells: Carefully transfer the cell suspension into a sterile centrifuge tube (ensuring the flow tube remains on the magnetic separator during transfer). The collected cell suspension contains the purified mouse neutrophils. Centrifuge at 500 × g for 5 min, discard the supernatant and collect the cells.

8. According to the experimental requirements, wash the cells and resuspend the cells in the appropriate buffer or medium for subsequent molecular biology or cell biology experiments.

1. During the use and storage of the Biotin-Antibody Mix, a void repeated freeze-thaw cycles, high-speed centrifugation, and other similar operations.

2. It is recommended to use low-adsorption consumables, such as pipette tips and centrifuge tubes to minimize loss of magnetic beads and antibodies due to adsorption.

3. During use and storage, magnetic beads should not be subjected to high-speed centrifugation, drying, or freezing. Avoid leaving magnetic beads in a magnetic field for extended periods, as this can cause bead aggregation, reducing their binding activity.

4. Before drawing magnetic beads from the storage tube, thoroughly mix them by gentle vortexing. During operation, handle gently and avoid creating air bubbles.

5. This pruduct should be used with a magnetic separator.

6. Perform all experimental procedures gently to avoid mechanical damage to the cells.

7. This product is for R&D use only, not for drug, household, or other uses.

8. For your safety and health, please wear a lab coat and disposable gloves to operate.