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Human CD4 T Cell Isolation Kit (Column-Free)

Human CD4 T Cell Isolation Kit (Column-Free)

Catalog Number: S0K3009 Reactivity: Human Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Human CD4 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-CD4 T cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-coated nanomagnetic beads. As a result, the non-target cells become bound to the magnetic beads via the antibody‑antigen complex, while the target CD4 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 CD4 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 CD4 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-free negative selection approach: non-CD4 T cells are labeled with biotin-conjugated antibodies, and unwanted cells are removed using magnetic bead separation, thereby enriching highly pure, high‑activity CD4 T cells. This method avoids the potential impact of direct labeling on CD4 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 provide it separately. The corresponding Staret product number isS0D3018

Steps

Operating Instructions

Dosage and timing

1

Peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood by density-gradient centrifugation using lymphocyte separation medium, or cryopreserved PBMCs are directly thawed and resuspended, followed by cell counting.

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

Prepare the sample

2

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

Note:Reagents are adjusted proportionally based on the sample volume; a pre-sorting sample is retained for determining the proportion of human CD4 T cells.

1×10sevencells/100 μL

3

Add 10 μL of the Human CD4 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 beads from settling excessively;

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) with 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;


Picture

Validation Data

Human CD4 T cells were enriched from peripheral blood mononuclear cells (PBMCs) and, after sorting, were stained with CD4-FITC (Clone: OKT4) and CD8-APC (Clone: SK1). Analysis was performed by gating on live cells (Live+). Flow cytometric results indicated that the purity of the sorted Human CD4 T cells was 95.3%.