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Human Naïve CD4 T Cell Isolation Kit (Column-Free)

Human Naïve CD4 T Cell Isolation Kit (Column-Free)

Catalog Number: S0K3014 Reactivity: Human Conjugation: Brand: Starter
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$1,000 USD
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Product Details

Product Specification


Format

1 mL Human Naïve CD4 T Cell Biotin-Antibody Cocktail, PBS buffer containing BSA;

1.5 mL Streptavidin Beads, PBS buffer containing BSA and Poloxamer 188;
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-human naïve CD4 T cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-nanomagnetic beads. As a result, the non-target cells are marked by the antibody‑bead complex, while the target human naïve 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 human naïve CD4 T cells remain in the supernatant. This supernatant constitutes the enriched fraction of human naïve CD4 T cells after removal of the non-target cells.
Applications in cell sorting Naïve CD4 T cells in their unmanipulated, label-free state are isolated from human PBMCs and can be used for a variety of downstream functional assays.
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

The immunophenotype of human naïve CD4 T cells is CD3⁺CD4⁺CD45RA⁺CD45RO⁻. They constitute the core precursor population of helper T cells in the human adaptive immune system, responsible for initial antigen recognition and directed differentiation, and are also a key cell subset for maintaining immune homeostasis, mediating specific responses, and establishing immunological memory. The human naïve CD4 T-cell negative-selection kit is developed based on immuno‑magnetic bead–mediated negative selection, overcoming the labeling limitations inherent in positive selection. By specifically depleting non‑target cells—such as CD8 T cells, NK cells, B cells, monocytes, dendritic cells, granulocytes, and memory T cells—it yields unlabeled, native‑state, highly purified naïve CD4 T cells, perfectly preserving their initial quiescent state, surface molecule integrity, and multipotential differentiation capacity. This makes them well suited for various functional assays, including in vitro activation and proliferation, subtype‑directed differentiation induction, and cytokine secretion analysis, and they have become the mainstream research tool for isolating human naïve CD4 T cells.

Protocol

Note: This kit does not include the sorting buffer; you will need to provide it yourself. The corresponding Stater product 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 400 g for 7 minutes;

Sample Preparation

2

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

Note:Adjust reagents proportionally according to sample volume; set aside a pre-sorting sample to determine the proportion of Human Naïve CD4 T cells.

1×10^77cells/100 μL

3

Add 10 μL of Human Naïve 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 evenly;

Mix for 5–30 seconds

6

Add 15 μL of Streptavidin beads to the sample;

15 μL/100 μL

7

Gently mix the magnetic beads and cells, and incubate at room temperature for 8 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 remix them again after about 2.5 minutes;

Incubate at room temperature for 8 minutes

8

Add MagSep Separation Buffer to the sample and immediately place it on the magnetic stand to prevent the magnetic beads from settling excessively;

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

Add the total volume to 7.5 mL (in a 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 400 g for 7 minutes, for subsequent culture and analysis.

Cell sorting successful

Note:1. 0.1-1 × 108cells (0.1-1 mL) are recommended to use a 5 mL single-well magnetic separator (Starter EasyIso Separator) with a total adsorption volume of 2.5 mL;

2-5 × 108For cells (2–5 mL), it is recommended to use a 15 mL multi-well magnetic separator (Starter EasyEights EasyIso Separator) The total adsorption volume is 7.5 mL;

2. If the sample viability is low, it is recommended to use a dead cell removal kit to eliminate dead cells (Dead Cell Removal Set, (RUO)#S0K0009);


Picture

Validation Data

Naïve CD4 T cells were enriched from human peripheral blood mononuclear cells (PBMCs) using a column-free sorting kit for Human Naïve CD4 T Cells. Following sorting, the cells were stained with fluorescent antibodies—CD3-FITC (Clone: UCHT1), CD4-Alexa Fluor® 647 (Clone: OKT4), CD45RA-PE-Cy7 (Clone: HI100), and CD45RO-Pacific Blue (Clone: UCHL1)—and analyzed by gating on live cells (Live⁺) to determine the proportion of Naïve CD4 T cells in the sorted fraction. Flow cytometric analysis revealed that, after sorting, the purity of CD45RA⁺CD45RO⁻ cells among CD3⁺CD4⁺ cells was 92.5%.