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

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

Catalog Number: S0K1002 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

This kit employs a column-based negative selection approach to isolate naïve CD8 T cells from mouse spleens. In this negative-selection protocol, non-naïve CD8 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 become bound to the magnetic beads via the antibody complexes, while the target naïve CD8 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 naïve CD8 T cells elute by gravity into the collection tube. The resulting supernatant represents the enriched fraction of naïve CD8 T cells after removal of the non-target cells.
Reactivity Mouse
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

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 exceeding 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 separation buffer; please prepare it yourself. The corresponding Stater catalog number isS0D3018

TakingL Separation Columnas an example

Steps

Operating instructions

Dosage and time

Cell processing and labeling

1

Take the mouse spleen, grind it on a 40 μm sterile filter to prepare a single-cell suspension of the spleen, perform red blood cell lysis, and then count the cells.

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

Note:Adjust reagents proportionally according to the sample volume; set aside a pre-sorting sample to determine 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, gentle pipetting to mix 2–3 times is recommended;

10 μL/100 μL

4

Incubate the antibodies in the cocktail with the cells at room temperature for 10 min;

Incubate at room temperature for 10 min

5

Add MagSep Separation Buffer equal to ten times the cell volume to wash the cells, centrifuge at 400g for 7 min, discard the supernatant, and resuspend to the original volume;

Centrifuge at 400g for 7 min

6

Mix wellStreptavidin Beads, add 20 μLStreptavidin Beadsto the sample;

20 μL/100 μL

7

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

Incubate at room temperature for 5 minutes

Cell sorting

8

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

Wash the separation column

9

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

10

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 has a maximum labeling capacity of1×108;

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-based sorting 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 assessed. Flow cytometric analysis revealed that the purity of the sorted Naïve CD8 T cells was 96.2%.