Human CD4+ T Cell Isolation Kit (negative selection)

1. Mouse Cells

2. Human Cells

1.High Purity: Efficient depletion of non-target cells enables the isolation of highly enriched target cells.
2.High Viability: Isolated cells maintain intact functional activity without abnormal activation, free of antibodies and magnetic beads
3.Easy Operation: Column-free separation using a simple magnetic rack.

Suitable for isolating CD4+ T cells from human PBMCs.

1.Human PBMC Preparation: Isolate PBMCs from human peripheral blood using Ficoll density gradient centrifugation. Wash the collected cells with PBS and centrifuge. Discard the supernatant, resuspend PBMCs in Isolation Buffer, and adjust cell concentration to 1 x 108 cells/mL.
Note: Recommended Isolation Buffer: PBS containing 2 mM EDTA and 2% FBS; or PBS containing 2 mM EDTA and 0.5% BSA. The buffer must be sterile-filtered through a 0.22 μm membrane filter before use.
2.Antibody Labeling: Add 100 μL cell suspension (1 x 107 cells) directly to the bottom of a sterile flow cytometry tube. Add 2 μL Biotin-Antibody Mix, mix thoroughly, and incubate at 4°C for 15 min. Add 10 volumes of Isolation Buffer, centrifuge at 500 x g for 5 min, and discard the supernatant. Resuspend cells in 100 μL Isolation Buffer.
Note: Avoid running cell suspensions down the tube wall when adding cells to the bottom. Scale up the Biotin-Antibody Mix volume proportionally when isolating more cells. Centrifuge tubes may also be used depending on the compatibility of the magnetic rack.
3.Bead Pretreatment: Resuspend magnetic beads by vortexing. Transfer the required amount of beads into a 1.5 mL tube, add 1 mL Isolation Buffer, centrifuge at 10,000 x g for 1 min, and discard the supernatant. Repeat the washing step once. Resuspend beads in Isolation Buffer back to their original volume. (e.g., if 20 μL beads were washed, resuspend in 20 μL Isolation Buffer after washing).
4.Magnetic Bead Labeling: Add 10 μL pretreated Streptavidin Magnetic Beads to the cell suspension from Step 2, mix well, and incubate at 4°C for 10 min.
Note: Scale up Streptavidin Magnetic Beads proportionally for larger cell numbers. For example, when isolating 5 x 10^7 cells, add 10 μL Biotin-Antibody Mix and 50 μL Streptavidin Magnetic Beads to 500 μL cell suspension. For ＜ 1 x 10^7 cells, adjust cell suspension volume to 100 μL, then add 2 μL Biotin-Antibody Mix and 10 μL Streptavidin Magnetic Beads.
5.Dilution: After incubation, add 2.5 mL Isolation Buffer to the flow tube and gently pipette up and down 5 times to mix. Avoid vigorous vortexing or tube inversion.
6.Magnetic Separation: Place the flow tube on a magnetic rack and let it stand for 5 min.
7.Cell Collection / First-Round Isolation: Gently pour the cell suspension containing purified CD4+ T cells into a sterile centrifuge tube while keeping the flow tube on the magnetic rack. The isolated cells are ready for downstream biological assays or flow cytometry analysis. To further increase purity, perform a second-round purification by centrifuging the cell suspension at 500 x g for 5 min, discarding the supernatant, resuspending in 100 μL Isolation Buffer, and executing the following steps.
Note: Scale up the resuspension volume accordingly for larger cell numbers. For example, when isolating 5 x 10^7 cells, resuspend the cell pellet in 500 μL Isolation Buffer after centrifugation.
8.Second-Round Bead Labeling: Add 10 μL pre-washed Streptavidin Magnetic Beads, mix gently, and incubate at 4°C for 10 min.
Note: Scale up Streptavidin Magnetic Beads proportionally for larger cell numbers. For example, when isolating 5 x 10^7 cells, add 50 μL Streptavidin Magnetic Beads to 500 μL cell suspension.
9.Second-Round Dilution: After incubation, top up Isolation Buffer to 2.5 mL, pipette gently 5 times to mix, and transfer the mixture into a sterile flow tube.
10.Second-Round Magnetic Separation: Place the flow tube on the magnetic rack and let it stand for 5 min.
11.Second-Round Cell Collection: Gently pour the cell suspension into a sterile tube while keeping the flow tube on the magnetic rack. The twice-purified human CD4+ T cells are collected.
12.Final Processing: Wash cells according to experimental requirements and resuspend them in desired buffer or culture medium for downstream molecular or cell biology experiments.

4°C, 2 years.

1.Do not freeze any kit components. Magnetic beads should be stored in their storage solution to prevent drying.
2.Resuspend the beads thoroughly by vortexing before pipetting from the stock tube, taking care to avoid air bubbles.
3.High-quality low-binding pipette tips and reaction tubes are recommended to prevent bead and solution loss due to wall adhesion.
4.This product is intended for scientific research purposes by professionals only and must not be used for clinical diagnosis or treatment, food, or medicine, nor stored in residential areas.
5.For your safety and health, please wear a lab coat and disposable gloves during operation.