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

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

Catalog Number: S0K3005 Reactivity: Mouse Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Mouse Naïve CD8 T Cell Biotin-Antibody Cocktail

2mL 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- is labeled with a biotin-conjugated antibody.Naïve CD8 TCells are then incubated with streptavidin‑coated nanomagnetic beads; non‑target cells are labeled via antibody‑bead complexes, while the target…Naïve CD8 TThe cells remain unlabeled. The cell suspension is then transferred to a polystyrene tube and placed within the sorter's magnetic field. Under the influence of the magnetic field, the magnetically labeled non-target cells adhere to the tube wall, while the unlabeledNaïve CD8 TThe cells remain in the supernatant. This supernatant is, therefore, the solution from which non-target cells have been removed.Naïve CD8 TCell-enriched fraction.
Reactivity Mouse
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

Mouse naïve CD8 T cells are cytotoxic T cells that have matured in the thymus but have not yet encountered specific antigens; they remain in a quiescent state. These cells primarily reside in peripheral lymph nodes and the spleen, where they rely on IL-7 signaling to maintain homeostatic survival. Upon activation by exogenous antigens and costimulatory signals, they undergo extensive clonal expansion and differentiate into effector CD8⁺ T cells (CTLs) with direct cytotoxic activity and long-lived memory CD8⁺ T cells. They serve as the pivotal initiating cells of the organism's adaptive immune responses against viruses and tumors. Using negative magnetic bead sorting to isolate and purify naïve CD8⁺ T cells from mouse splenic cell suspensions yields a purity of over 90%. The sorted cells bear no surface antibody labeling and retain their natural quiescent state, making them suitable for subsequent in vitro activation, differentiation, and functional studies.

Protocol

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

Steps

Operating Instructions

Dosage and Time

1

Collect the mouse spleen, grind it on a 40 μm sterile filter to prepare a single-cell suspension of splenic cells, and perform cell counting;

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

Prepare the sample

2

Resuspend 1×10 in 100 μL of MagSep Separation Buffer7cells;

Note:Adjust reagents proportionally according to the sample volume; set aside a pre-sorting sample to measure the initial naïve CD8 ratio;

1×107cells/100 μL

3

Add 10 μL of Mouse Naïve CD8 T cell Biotin Antibody Cocktail to the sample and gently mix.

Note:For 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 by shaking for 5–30 seconds

6

Add 20 μL of Streptavidin beads to the sample;

20 μL/100 μL

7

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

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

During this step, the magnetic beads may settle at the bottom; you can gently tap the tube to remix them around the 2.5-minute mark;

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 (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 400g 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 volume of 2.5 mL;

2-5 × 108For cells (2–5 mL), it is recommended to use a 15 mL 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 a dead cell removal kit to eliminate dead cells (Dead Cell Removal Set, (RUO)#S0K0009).


Picture

Validation Data

Naïve CD8 T cells were enriched from mouse splenocytes using a column-free Naïve CD8 T cell isolation kit. Cells were stained with the following fluorescent antibodies: CD8-Pacific Blue (SDT Cat: S0B8086), CD44-FITC (SDT Cat: S0B5452), and CD62L-Alexa Fluor® 647 (SDT Cat: S0B1699). Gating was performed on live cells (Live+), and the proportion of Naïve CD8 T cells (CD8+CD44-CD62L+) in the sorted fraction was determined. Flow cytometry analysis revealed that the purity of the isolated Naïve CD8 T cells was 96.1%.