Human CD3+T Cell Sorting Kit (Negative Selection)

Cat. No.: H1522404

GRADE & PURITY BioReagent

Overview

The Human CD3⁺ T Cell Isolation Kit (Negative Isolation) is used to isolate CD3⁺ T cells from human peripheral blood mononuclear cells (PBMCs) by negative i solation. The principle is to use a cocktail of biotin-labeled monoclonal antibodies against non-target cells (non-CD3⁺ T cells), followed by removal of these non-target cells using streptavidin-labeled magnetic beads, thereby achieving the isolation of human peripheral blood CD3⁺ T cells. A magnetic separator is required for the isolation process.

Applications

Isolation of CD3⁺ T cells from human PBMCs.

H1522404	Component	Appearance	10T	50T	100T	Storage
H1522404A	Biotin-Antibody Mix	Liquid	20 μL	100 μL	200 μL	2-8℃.
H1522404B	Streptavidin Magnetic Beads	Liquid	200 μL	1 mL	1 mL×2	2-8℃.

Protocol Example

1. Prepare human PBMCs

   Isolate PBMCs from human peripheral blood using Ficoll density gradient centrifugation. Collect the PBMCs, wash the cells with PBS, and after centrifugation, resuspend the PBMCs 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.

2. 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: Add the cell suspension directly to the bottom of the FACS 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.

3. Add Streptavidin Magnetic Beads

   After incubation, add 20 μL of washed Streptavidin Magnetic Beads to the FACS tube, mix well, and incubate at 4°C for 10 min (the beads must be washed before use: vortex thoroughly to resuspend the beads, transfer the required volume of beads to a 1.5 mL centrifuge tube, add Isolation Buffer to a total volume of 1 mL, 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).

   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.

4. Dilute and mix

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

5. Magnetic separation

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

6. Collect CD3⁺ 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 human CD3⁺ T cells. Centrifuge at 300 g for 5 min, then discard the supernatant and collect the cells.

7. 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

CD3⁺ T cells were isolated from human PBMCs. Cells before and after isolation were stained with PE-labeled anti-human CD3 antibody (clone OKT3) and analyzed by flow cytometry. The purity of CD3⁺ T cells before and after isolation was 69.1% and 97.9%, 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.

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

H1522404	Component	Appearance	10T	50T	100T	Storage
H1522404A	Biotin-Antibody Mix	Liquid	20 μL	100 μL	200 μL	2-8℃.
H1522404B	Streptavidin Magnetic Beads	Liquid	200 μL	1 mL	1 mL×2	2-8℃.