Mouse CD4⁺ T Cell Isolation Kit (Negative Isolation)

Cat. No.: M1522407

GRADE & PURITY BioReagent

Overview

The Mouse CD4⁺ T Cell Isolation Kit (Negative Isolation) is used to isolate CD4⁺ T cells from mouse spleen or other tissue single-cell suspensions by negative i solation. The principle is to use a cocktail of biotin-labeled monoclonal antibodies against non-target cells (non-CD4⁺ T cells), followed by removal of these non-target cells using streptavidin-labeled magnetic beads, thereby achieving the isolation of mouse CD4⁺ T cells. A magnetic separator is required for the isolation process.

Applications

Isolation of CD4⁺ T cells from mouse spleen and lymph nodes.

M1522407	Component	Appearance	10T	50T	100T	Storage
M1522407A	Biotin-Antibody Mix	Liquid	20 μL	100 μL	200 μL	2-8℃.
M1522407B	Streptavidin Magnetic Beads	Liquid	200 μL	1 mL	1 mL×2	2-8℃.

Protocol (Using Mouse Spleen CD4⁺ T Cell Isolation as an Example)

1. Prepare single-cell suspension

   Grind the spleen on a 70 μm cell strainer, rinse the strainer with pre-chilled PBS, collect the cell suspension into a 50 mL centrifuge tube, and centrifuge at 500 g for 5 min.

2. Lyse red blood cells

   After centrifugation, discard the supernatant, add 5 mL of ACK red blood cell lysis buffer, lyse at room temperature for 5 min, then add 20 mL PBS and centrifuge at 500 g for 5 min.

   Note: The red blood cell lysis step can be adjusted in volume and time depending on the lysis buffer used. A small amount of red blood cell residue will not affect subsequent isolation or cell purity.

3. Filter and count cells

   After centrifugation, discard the supernatant, resuspend the splenocytes in PBS, filter the cell suspension through a 70 μm cell strainer, and count the cells. After counting, centrifuge at 500 g for 5 min.

   Note: The cell suspension must be passed through a cell strainer to remove tissue and cell clumps; otherwise, subsequent cell isolation purity may be affected.

4. Adjust cell density

   After centrifugation, discard the supernatant, resuspend the cells in Isolation Buffer to a density of 1×10⁸ cells/mL.

   Note: Isolation buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS), or PBS containing 2 mM EDTA and 0.5% BSA. The buffer must be pre-filtered through a 0.22 μm filter.

5. Add Biotin-Antibody Mix

   Add 100 μL of cell suspension (1×10⁷ cells) to the bottom of a sterile FACS tube, then add 2 μL of Biotin-Antibody Mix. Mix well and incubate at 4°C for 10 min.

   Note:

   • When adding the cell suspension, add it to the bottom of the tube, avoiding the tube wall.

   • Depending on the magnetic separator used, a centrifuge tube can also be used for cell isolation.

   • If isolating more cells, increase the volume of Biotin-Antibody Mix proportionally.

6. Add Streptavidin Magnetic Beads

   After incubation, add 20 μL of washed Streptavidin Magnetic Beads to the FACS tube (the beads must be washed before use: vortex to resuspend the beads, transfer the required volume of beads to a 1.5 mL centrifuge tube, add 1 mL of Isolation Buffer, centrifuge at 10,000 g for 1 min or use a magnetic separator for 3 min, then discard the supernatant. Add 1 mL of Isolation Buffer and wash the beads once more, then resuspend the beads in the same volume of Isolation Buffer as originally taken. For example, if 20 μL of beads are taken, resuspend them in 20 μL of Isolation Buffer after washing). Mix well and incubate at 4°C for 10 min.

   Note:

   • If isolating more cells, increase the volume of Streptavidin Magnetic Beads proportionally. For example, to isolate 5×10⁷ cells, add 10 μL of Biotin-Antibody Mix and 100 μL of Streptavidin Magnetic Beads to 500 μL of cell suspension.

   • If isolating fewer than 1×10⁷ cells, adjust the cell suspension volume to 100 μL, then add 2 μL of Biotin-Antibody Mix and 20 μL of Streptavidin Magnetic Beads.

7. Dilute and mix

   After incubation, add 2.5 mL of Isolation Buffer to the FACS tube, and mix gently by pipetting up and down 5 times (avoid vigorous shaking or inverting).

8. Magnetic separation

   Place the FACS tube containing the cell suspension on a magnetic separator and let stand for 5 min.

9. Collect CD4⁺ T cells

   Gently pour the cell suspension into a sterile centrifuge tube (do not remove the FACS tube from the magnetic separator while pouring). This cell suspension contains the purified mouse CD4⁺ T cells. Centrifuge at 500 g for 5 min, then discard the supernatant and collect the cells.

10. Resuspend for further use

    Wash the cells as required for your experiment, then resuspend them in the desired buffer or culture medium for subsequent molecular or cell biology experiments.

Isolation Performance

CD4⁺ T cells were isolated from C57BL/6 mouse splenocytes. Cells before and after isolation were stained with FITC anti-mouse CD4 antibody (clone GK1.5) and analyzed by flow cytometry. The purity of CD4⁺ T cells before and after isolation was 20.3% and 98.8%, respectively.

Precautions

1. Avoid freezing the magnetic beads and antibody mix during use and storage.

2. Low-retention pipette tips and centrifuge tubes are recommended to avoid loss of beads and antibodies due to adsorption.

3. This product must be used with a magnetic separator.

4. For research use only.

Specifications

Specifications & Purity

BioReagent

Grade

BioReagent

Application

Cell analysis

Storage and Shipping

Storage

Store at 2-8°C,Do not freeze

Shipped In

Wet ice,Do not freeze

Stability And Storage

Store at 2-8℃ long term (12 months). Do not freeze.

Contents & Storage

M1522407	Component	Appearance	10T	50T	100T	Storage
M1522407A	Biotin-Antibody Mix	Liquid	20 μL	100 μL	200 μL	2-8℃.
M1522407B	Streptavidin Magnetic Beads	Liquid	200 μL	1 mL	1 mL×2	2-8℃.