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Human T Cell Isolation Kit (Column-Free)

Human T Cell Isolation Kit (Column-Free)

Catalog Number: S0K3002 Reactivity: Human Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Human T Cell Biotin-Antibody Cocktail

1mL Streptavidin Beads
Capacity

Suitable for a total cell count of 1×10⁹, and can perform up to 100 sorting runs.
Principle of Separation

In this negative selection process, non-T cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin‑nanomagnetic beads. As a result, the unwanted cells become bound to the magnetic beads via the antibody complex, while the target T cells remain unlabeled. Subsequently, the cell suspension is transferred to a polystyrene tube and placed within the magnetic field of the sorter. Under the influence of the magnetic field, the magnetically labeled non‑target cells adhere to the tube wall, whereas the unlabeled T cells remain in the supernatant. This supernatant constitutes the T‑cell‑enriched fraction after removal of the non‑target cells.
Reactivity Human
Magnetic Bead Size 150 nm
Separation Method Column-Free
Selection Strategy Negative Selection
Stability & Storage

Store protected from light at 2–8°C; do not freeze.

Background

Human T cells are key immune cells in the immune system, playing a central role in immune responses, immune regulation, and defense against infection and tumors. This kit employs a column-based negative selection approach: non-T cells are labeled with biotin-conjugated antibodies, and unwanted cells are removed using magnetic bead separation, thereby enriching highly pure, high‑activity T cells. This method avoids the potential impact of direct labeling on T cell function and is suitable for downstream experimental studies such as cell culture and functional assays.

Protocol

Note: This kit does not include sorting buffer; please prepare it yourself. The corresponding Stater catalog number isS0D3018

Steps

Operating Instructions

Dosage and Time

1

Isolate PBMCs from peripheral blood using density gradient centrifugation with lymphocyte separation solution, or directly thaw cryopreserved PBMC cells, and proceed with cell counting;

Note: It is recommended to count cells after staining with AO/PI; PBMCs can be centrifuged at 400g for 7 minutes;

Sample Preparation

2

Resuspend 1×10 in 100 μL of MagSep Separation Buffer.7Cells;

Note:Adjust the reagents proportionally according to the sample volume; set aside a pre-sorting sample to test the proportion of human T cells;

1×107cells/100 μL

3

Add 10 μL of Human T cell Biotin Antibody Cocktail to the sample and gently mix.

Note:In this step, gently pipette up and down 2–3 times to mix thoroughly;

10 μL/100 μL

4

Incubate the antibodies in the cocktail with the cells at room temperature for 10 minutes;

Incubate at room temperature for 10 minutes

5

Shake for 5–30 seconds to mix the Streptavidin beads;

Shake for 5–30 seconds

6

Add 10 μL of Streptavidin beads to the sample;

10 μL/100 μL

7

Gently mix the magnetic beads and cells, and incubate at room temperature for 5 minutes;

Note:For mixing in this step, you can use a pipette to gently blow the mixture 2–3 times;

During this step, the magnetic beads may settle at the bottom; you can mix them again after about 2.5 minutes;

Incubate at room temperature for 5 minutes

8

Add MagSep Separation Buffer to the sample, and immediately place it on the magnetic stand to prevent the magnetic beads from settling heavily at the bottom;

Add the total volume to 2.5 mL (5 mL flow cytometry tube)

Add the total volume to 7.5 mL (15 mL centrifuge tube)

9

Place the sample on the magnetic stand so that the magnetic beads adhere to the tube wall;

Adsorb using a single-well magnetic stand (3 min)

Adsorb using a multi-well magnetic stand (5 min)

10

Tilt the magnetic stand and pour the sample into a new collection tube to collect the sample;

Note:The sorted cells can be centrifuged at 400g for 7 minutes, for subsequent culture and analysis.

Cell sorting successful

Note:For 0.1–1 × 10^8 cells (0.1–1 mL), it is recommended to use a 5 mL single-well magnetic separator (Starter EasyIso Separator) to adsorb a total volume of 2.5 mL;

2–5 × 10^8 cells (2–5 mL) are recommended to be adsorbed using a 15 mL row-type magnetic separator (Starter EasyEights EasyIso Separator), with a total volume of 7.5 mL;


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Validation Data

Human T cells were enriched from peripheral blood mononuclear cells (PBMCs). After fluorescent staining with CD3-Alexa Fluor® 488 (SDT Cat: S0B1636), live cells were gated for analysis. Flow cytometry results showed that the purity of the sorted human T cells was 96%.