Mouse CD8+ Cell Isolation Kit (positive selection)

Mouse Cells

Human Cells

|Catalog No.|Product Name|Packing (for 1×10^9 cells)| |C0068-1|CD8 Capture Antibody|200 μL| |C0068-2|Releasable Magnetic Beads|2 mL| |C0068-3|Magnetic Beads Release Buffer|40mL|

1.High Purity: Can yield Isolated cells with a purity exceeding 95%.

2.Broad Compatibility: Target cells can be directly captured from PBMCs or whole blood.

3.Bead-release Technology: Can yield bead-free cells that are not affected in downstream applications.

Suitable for isolating CD8+ cells from mouse lymphoid organs or other tissues, such as spleen and lymph nodes.

Example: Isolation of CD8+ Cells from Mouse Spleen

1.To prepare a single-cell suspension: Place the mouse spleen on a 70 μm cell strainer and gently grind. Rinse the strainer with pre-cooled PBS to collect the cell suspension into a 50 mL centrifuge tube. Centrifuge at 500 g for 5 minutes.

2.Discard the supernatant after centrifugation. Add 5 mL of ACK lysing buffer and incubate at room temperature for 5 minutes. Add 20 mL of PBS, centrifuge at 500 g for 5 minutes.

Note: The lysis time and volume may be adjusted based on the buffer used. A small number of residual red blood cells will not significantly affect downstream separation or purity.

3.After centrifugation, discard the supernatant and resuspend the splenocytes in PBS. Filter the suspension through a 70 μm strainer. Count the cells and centrifuge again at 500 g for 5 minutes.

Note: To avoid debris and cell clumps that may affect sorting purity, ensure the suspension is filtered through a cell strainer.

4.After centrifugation, discard the supernatant and resuspend the cells in isolation buffer. Adjust the cell concentration to 1×10^8 cells/mL.

Note: Recommended isolation buffer: a. PBS（2 mM EDTA and 2% FBS）; b. PBS（2 mM EDTA and 0.5% BSA）. The buffer should be pre-filtered using a 0.22 μm membrane for sterilization.

5.Transfer 500 μL of the cell suspension (5×10^7 cells) to the bottom of a sterile flow tube. Add 10 μL of CD8 Capture Antibody, mix thoroughly, and incubate at 4°C for 15 minutes.

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, proportionally increase the volume of CD8 Capture Antibody used. Depending on the magnetic separator used, centrifuge tubes may also be suitable.

6.To prepare the magnetic beads: Vortex to resuspend the beads. Transfer the required amount of beads to a 1.5 mL centrifuge tube. Add 1 mL of isolation buffer, and centrifuge at 10,000 g for 1 minute. Discard the supernatant and repeat the washing step once. The volume of isolation buffer used for beads resuspension should be equal to the initial volume of beads that was aspirated. E.g., if 20 μL of beads are used for washing, resuspend in 20 μL of isolation buffer.

7.After cell incubation, add 100 μL of pre-treated Releasable Magnetic Beads to the tube, mix well and incubate at 4°C for 15 min.

Note: If a larger number of cells need to be isolated, the amount of Releasable Magnetic Beads can be increased proportionally. If isolating less than 1×10^7 cells, adjust the volume of cell suspension to 100 μL and add 2 μL of CD8 Capture Antibody along with 20 μL of Releasable Magnetic Beads.

8.After incubation, add 2.5 mL of isolation buffer to the tube and mix 5 times gently (avoid vigorous shaking or up-and-down mixing). Place the tube in the magnetic separator for 5 minute.

9.Discard the supernatant. Remove the flow tube from the magnetic separator and immediately add 2 mL of isolation buffer. Gently pipette up and down several times to fully resuspend the magnetic beads. Then place the tube back on the magnetic separator and let it stand for 5 minutes.

10.Repeat the washing step twice.

Note: Thorough washing helps ensure high purity of the target cells during the subsequent bead release process.

11.After magnetic separation, discard the supernatant. Remove the flow tube from the magnetic separator and immediately add 1 mL of Magnetic Beads Release Buffer to resuspend the beads, ensuring they do not dry out. Transfer the bead suspension to a 1.5 mL centrifuge tube and incubate with gentle rotation at room temperature for 10 minutes.

Note: The volume of Magnetic Beads Release Buffer can be adjusted according to the number of cells being processed. If fewer than 1×10⁷ cells are being sorted, use 200 μL of Magnetic Beads Release Buffer for elution.

12.After incubation, pipette the bead suspension up and down at least 10 times, then transfer it to a new flow tube. Add isolation buffer to a final volume of 2.5 mL, gently pipette to mix, and place the tube on the magnetic separator for 5 minutes.

13.The supernatant now contains the target cells. Transfer the supernatant to a 15 mL centrifuge tube and set aside. Immediately add 1 mL of Magnetic Beads Release Buffer to resuspend the beads, ensuring they do not dry out. Transfer the suspension to a 1.5 mL centrifuge tube. Incubate with gentle rotation at room temperature for 10 minutes.

14.Repeat the elution process (Step 12) once more and collect the supernatant.

15.Combine the supernatants from both elution steps and centrifuge at 500 g for 5 minutes. Discard the supernatant to obtain bead-free CD8+ cells.

16.Wash cells according to the requirements of experiment, and resuspend the cells in the appropriate buffer or medium. The cells can be used for downstream molecular or cell biology experiments.

Store at 4℃ for 2 years

1.Avoid freezing. Store beads in the solution to prevent drying.

2.Before removing beads from the tube, ensure they are evenly suspended by gentle shaking. Handle gently to prevent the bubbles.

3.It is recommended to use high-quality pipette tips and centrifuge tubes to avoid loss of beads due to adhesion.

4.This product is for scientific research use by professionals only and must not be used for clinical diagnosis or treatment, food or drug applications, and must not be stored in residential areas.

5.For your safety and health, wear lab coats and disposable gloves during operation.