Beskrivelse
The Human CD8+ T Cell Isolation Kit (Negative Selection) isolates and purifies CD8+ T cells from human peripheral blood mononuclear cells (PBMCs) via negative selection. The principle involves using a biotin-labeled monoclonal antibody cocktail to label non-target cells (non-CD8+ T cells), followed by the removal of these non-target cells using streptavidin-labeled magnetic beads, thereby obtaining unlabeled human CD8+ T cells.
Product Features
Simple Operation: No separation column is required; target cell separation can be achieved using a magnetic separator.
High Purity: The purity of isolated cells can reach over 90%.
Good Viability: As a negative selection method, magnetic beads do not directly contact the cells. There is no abnormal activation after sorting, ensuring downstream experiments are unaffected.
Fast Speed: Sorting can be completed in as little as 30 minutes.
Specifications
|
Cat.No. |
37659ES10 / 37659ES50 / 37659ES60 |
|
Size |
10 T / 50 T / 100 T |
Components
|
Cat. No. |
Component Name |
37659ES10 (10 T) Separation Capacity: Up to 1×10⁸ cells |
37659ES50 (50 T) Separation Capacity: Up to 5×10⁸ cells |
37659ES60 (100 T) Separation Capacity: Up to 1×10⁹ cells |
|
37659-A |
Human CD8+ T Cell Negative Antibody Cocktail |
20 μL |
100 μL |
200 μL |
|
37659-B |
Streptavidin Magnetic Beads |
0.2 mL |
1 mL |
2 mL |
Storage
Store at 2~8°C, valid for 1 year. Do not freeze.
Instructions for Use
1. Preparation of Required Materials
1) Separation Buffer: PBS containing 2 mM EDTA and 2% Fetal Bovine Serum (FBS) or PBS containing 2 mM EDTA and 0.5% BSA, which needs to be filter-sterilized through a 0.22 µm filter membrane in advance.
2) Sorting Materials and Separator: Sterile flow cytometry tubes or centrifuge tubes, magnetic separator.
2. Cell Sorting, using the sorting of CD8+ T cells from human PBMCs as an example:
1) Preparation of single-cell suspension: Isolate PBMCs from peripheral blood using Ficoll density gradient centrifugation, collect PBMCs, wash the cells with PBS, and after centrifugation, resuspend the PBMCs in the separation buffer. Adjust the cell density to 1×10⁸ cells/mL.
2) Aspirate 100 µL of cell suspension (1×10⁷ cells) into the bottom of a sterile 1.5 mL centrifuge tube, then add 2 µL of Human CD8+T Cell Negative Antibody Cocktail (37659-A). Mix well and incubate at 4°C for 15 min. Add 10 volumes of separation buffer, centrifuge at 500 g for 5 min, and discard the supernatant. Resuspend the cells with 100 µL of separation buffer.
[Note]: If sorting more cells, increase the amount of Human CD8+T Cell Negative Antibody Cocktail (37659-A) and separation buffer proportionally. You can perform the operation using 15 mL or 50 mL centrifuge tubes.
For example, when sorting 5×107cells, add 10 μ L Human CD8+ T Cell Negative Antibody Cocktail (37659-A) to 500 μ L cell suspension, wash with 5 mL separation buffer, and resuspend the cells in 500 μ L separation buffer after centrifugation.
3) Add 10 μ L of washed Streptavidin Magnetic Beads (37659-B) to the centrifuge tube, mix well, and incubate at 4°C for 10 min.
[Note]: Magnetic beads must be washed with separation buffer before use: Vortex to resuspend the magnetic beads, transfer the required amount of magnetic beads for the experiment to a 1.5 mL centrifuge tube, add 1 mL separation buffer, centrifuge at 10000 g for 1 min, and discard the supernatant. Add 1 mL separation buffer to repeat the washing of the magnetic beads once, then resuspend the magnetic beads in the same volume of separation buffer as the original. For example, if 20 \μ L of magnetic beads are taken for washing, resuspend in 20 \μ L separation buffer after washing.
If sorting more cells, increase the amount of Streptavidin Magnetic Beads (37659-B) proportionally.
If sorting less than 1×107 cells, adjust the cell suspension volume to 100 μ L, add 2 μ L Human CD8+ T Cell Negative Antibody Cocktail (37659-A) and 10 μ L Streptavidin Magnetic Beads (37659-B).
4) After incubation, transfer the cell and magnetic bead mixture to 1 sterile flow cytometry tube, bring the volume to 2.5 mL with separation buffer, and mix gently by pipetting up and down 5 times with a pipette (avoid vigorous shaking or inverting up and down to mix).
5) Place the sorting flow cytometry tube containing cells on a magnetic rack and let stand for 5 min.
6) Gently pour the cell suspension into 1 sterile centrifuge tube (do not remove the flow cytometry tube from the magnetic rack during pouring). This cell suspension contains the purified human CD8+ T cells, which can be used for subsequent molecular biology or flow cytometry detection. If further improvement of CD8+ T cell purity is required, centrifuge the cell suspension at 500 g for 5 min, discard the supernatant, resuspend the cells in 100 μ L separation buffer, and continue with the secondary purification according to the following steps.
[Note]: If sorting more cells, increase the resuspension volume accordingly. For example, when sorting 5×107 cells, resuspend in 500 μ L separation buffer after centrifugation.
7) Add 10 μ L of washed Streptavidin Magnetic Beads (37659-B) to the centrifuge tube, mix well, and incubate at 4°C for 10 min.
Note: If sorting more cells, increase the amount of Streptavidin Magnetic Beads (37659-B) proportionally.
8) After incubation, bring the volume to 2.5 mL with separation buffer, mix gently by pipetting up and down 5 times with a pipette, and transfer the cell and magnetic bead mixture to 1 sterile flow cytometry tube.
9) Place the sorting flow cytometry tube containing cells on a magnetic rack and let stand for 5 min.
10) Gently pour the cell suspension into 1 sterile centrifuge tube (do not remove the flow cytometry tube from the magnetic rack during pouring). This cell suspension contains the secondary purified human CD8+ T cells. The secondary purification can increase the purity of CD8+ T cells by 2-4%.
11) After washing the cells according to experimental needs, resuspend the cells in the required buffer or culture medium, and they can be used for subsequent molecular biology or cell biology experiments.
Notes
1. Avoid freezing the Negative Antibody Cocktail and Streptavidin Magnetic Beads during use and storage.
2. It is recommended to use low-adsorption centrifuge tubes and pipette tips to avoid the loss of magnetic beads and antibodies due to adsorption.
3. This product must be used in conjunction with a magnetic separator.
4. This product is for research use only!
5. For your safety and health, please wear a lab coat and disposable gloves when handling.
Documents:
Safety Data Sheet
Manuals:
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