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Human Naïve CD8 T Cell Isolation Kit (Column-Free)

Human Naïve CD8 T Cell Isolation Kit (Column-Free)

Catalog Number: S0K3019 Reactivity: Human Conjugation: Brand: Starter
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$1,250 USD
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Product Details

Product Specification


Format

1 mL Human Naïve CD8 T Cell Biotin-Antibody Cocktail;

1.5 mL Streptavidin Beads;

All components are stored in a buffer containing 0.05% sodium azide.
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 CD8 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 are marked by the antibody-bead complex, while the target human naïve CD8 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 CD8 T cells remain in the supernatant. This supernatant constitutes the enriched fraction of human naïve CD8 T cells after removal of the non-target cells.
Applications in cell sorting Naïve CD8 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

Human naive CD8⁺ T cells are the core initiating cell population of the adaptive immune response, referring to a subset of CD8⁺ T cells that have not encountered specific antigens and remain in a quiescent state, typically characterized by the phenotype CD8⁺CD45RO⁻CD45RA⁺CCR7⁺. Upon antigen stimulation and cytokine regulation, these cells can be activated and proliferate, differentiating into effector and memory CD8⁺ T cells. They play a crucial role in research on anti-infectious immunity, tumor immunity, and autoimmune diseases, making them an important subject of study in the field of adaptive immunity. The Human Naive CD8⁺ T Cell Negative Selection Kit is developed based on immunomagnetic bead–mediated negative selection technology, which avoids the antibody-binding labeling defects inherent in positive selection. The antibody components in the separation system precisely target surface markers on non-naive CD8⁺ T cells, specifically depleting non-target cell populations such as CD4⁺ T cells, memory CD8⁺ T cells, B cells, NK cells, monocytes, granulocytes, and erythrocytes. The resulting high-purity human naive CD8⁺ T cells are free from exogenous labels and magnetic bead residues, maintaining their native state and preserving their biological potential for proliferation and differentiation to the greatest extent. They are fully compatible with various in vitro functional assays and represent the mainstream tool for isolating and purifying human naive CD8⁺ T cells in the research community today.

Protocol

Note: This kit does not include the sorting buffer; it must be provided separately. The corresponding Stater product number is S0D3018.

Steps

Operating instructions

Dosage and time

1

Isolate PBMCs from peripheral blood using density gradient centrifugation with lymphocyte separation medium, or directly thaw cryopreserved PBMCs, and perform 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 1×10⁷cells in 100 μL of MagSep Separation Buffer;

Note:Adjust reagent volumes proportionally based on sample volume; retain a pre-sorting sample to determine the proportion of human naïve CD8 T cells;

1×10⁷cells/100 μL

3

Add 10 μL of Human Naïve CD8 T Cell Biotin-Antibody Cocktail to the sample and gently mix.

Note:In this step, the mixture can be gently mixed by pipetting up and down 2–3 times.

10 μL/100 μL

4

Incubate the antibodies and cells in the cocktail at 4°C or room temperature for 10 minutes.

Incubate at 4°C or at room temperature for 10 minutes.

5

After antibody incubation, add twice the volume of MagSep Separation Buffer, centrifuge at 400 g for 7 minutes, and then carefully discard the supernatant. Resuspend 1×10⁷ in 100 μL of MagSep Separation Buffer.Cells, proceed to the next step;

Add a buffer volume twice the size to wash the antibody.

6

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

Mix thoroughly for 5–30 seconds.

7

Add 15 μL of streptavidin beads to the sample.

15 μL/100 μL

8

Gently mix the magnetic beads and cells, and incubate at 4°C or room temperature for 8 minutes.

Note:In this step, you can gently pipette the mixture 2-3 times to mix it evenly.

In this step, the magnetic beads may settle at the bottom. You can mix them again after 4 minutes.

Incubate at 4°C or room temperature for 8 minutes.

9

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.

Adjust the total volume to 2.5 mL (using a 5 mL flow cytometry tube).

Adjust the total volume to 7.5 mL (using a 15 mL centrifuge tube).

10

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

Single-well magnetic stand adsorption (3 min).

Multi-well magnetic stand adsorption (5 min).

11

Tilt the magnetic stand and pour the sample into a new adsorption tube. Repeat step 10 to perform a second adsorption of the magnetic beads, thereby improving the purity of the separation.

Single-well magnetic stand adsorption (3 min).

Multi-well magnetic stand adsorption (5 min).

12

Tilt the magnetic rack 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 and used for subsequent culture and analysis.

Cell sorting successful.

Note:1. 0.1-1×10⁸For 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 for adsorption.

2-5×10⁸For cells (2–5 mL), it is recommended to use a 15 mL multi-tube magnetic rack (Starter EasyEights EasyIso Separator) with a total adsorption volume of 7.5 mL.

2. If the sample viability is low, it is recommended to use the Dead Cell Removal Set (RUO) #S0K0009 to remove dead cells.


Picture

Validation Data

Human naïve CD8 T cells were enriched from peripheral blood mononuclear cells (PBMCs). After sorting, the cells were stained with the following fluorescent antibodies: CD8-Pacific Blue (Clone: S-R533), CD45RO-PE-Cy7 (Clone: UCHL1), CD45RA-APC (Clone: HI100), and CCR7 (CD197)-FITC (Clone: S-R704). Gating was performed on live cells (Live⁺) for analysis, and the proportion of naïve CD8 T cells in the sorted fraction was determined. Flow cytometry results showed that the purity of CD8⁺CD45RO⁻ cells after sorting was 93.5%, and the purity of CD45RA⁺CCR7⁺ cells within the CD8⁺CD45RO⁻ population was 99.1%.