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

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

Catalog Number: S0K1014 Reactivity: Human Conjugation: Brand: Starter
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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

This kit employs a column-based negative selection approach to isolate Human Naïve CD4 T cells from human peripheral blood mononuclear cells (PBMCs). 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 unwanted cells become bound to the magnetic beads via the antibody‑bead complex, while the target Human Naïve CD4 T cells remain unlabeled. The cell suspension is subsequently loaded onto the separation column; under the influence of a magnetic field, the magnetically labeled non-target cells are retained within the column, whereas the unlabeled Human Naïve CD4 T cells elute by gravity into the collection tube. The resulting supernatant represents 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 20 nm
Separation Method Column-Based
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 methods. 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 suitable 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 sorting buffer; please provide it separately. The corresponding Staret product number isS0D3018

withL Separation ColumnFor example

Steps

Operating Instructions

Dosage and timing

Cell processing and labeling

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 pelleted by centrifugation at 400 g 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 assessing the proportion of human naïve CD4 T cells.

1×10sevencells/100 μL

3

Add 10 μL of the 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 min;

Note:If you want a higher cell yield, after antibody incubation, add twice the volume of MagSep Separation Buffer, centrifuge at 400 g for 7 min, then carefully discard the supernatant. Resuspend 1×10 in 100 μL of MagSep Separation Buffer.7cells, and proceed to the next steps.

Incubate at room temperature for 10 min

5

Mix wellStreptavidin Beads, add 15 μLStreptavidin Beadsto the sample;

15 μL/100 μL

6

Gently mix the magnetic beads and cells, and incubate at room temperature for 8 minutes;

Incubate at room temperature for 8 minutes

Cell sorting

7

In the column separation magnet, installL Separation Column, add 3 mL of MagSep Separation Buffer to wash the separation column;

Wash the separation column

8

Add the cells to the separation column and collect the target cells using a collection tube;

Note:The cell volume should be at least 500 μL; if insufficient, make up the volume with MagSep Separation Buffer; the cells that drip down under gravity are the target cells;

Collect the target cells

9

Add 3 mL of MagSep Separation Buffer and collect the target cells;

Note: This step elutes the unlabeled target cells from the separation column, increasing the yield.

Collect the target cells

Notes: 1. Pay attention to the maximum capacity of the separation column,The S Separation Column can label a maximum of1×107cells;The L Separation Column can label a maximum of1×108cells;

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

Human naïve CD4 T cells were enriched from peripheral blood mononuclear cells (PBMCs). Following sorting, the cells were stained with the fluorescent antibodies CD3-FITC (Clone: UCHT1), CD4-Alexa Fluor® 647 (Clone: OKT4), CD45RA-PE-Cy7 (Clone: HI100), and CD45RO-Pacific Blue (Clone: UCHL1). Gating was performed on live cells (Live⁺) for analysis, and the proportion of naïve CD4 T cells in the sorted fraction was determined. Flow cytometric analysis revealed that, after sorting, the purity of CD45RA⁺CD45RO⁻ cells among CD3⁺CD4⁺ cells was 98.8%.