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Isolate untouched mouse CD8+ T cells by depleting non-CD8+ T cells (CD4+ T cells, B cells, monocytes/ macrophages NK cells, dendritic cells, erythrocytes and granulocytes) from mouse spleen or lymph node cells. Other sources of mouse CD8+ T cells can also be used as starting material after optimization for the particular application. Isolated CD8+ T cells are bead- and antibody-free and are suitable for any downstream application
Principle of Isolation
Add a mixture of monoclonal antibodies against the non-CD8+ T cells to the starting sample. Add Mouse Depletion Dynabeads to bind to the non-CD8+ T cells during a short incubation. Separate the bead-bound cells by a magnet. Discard the beadbound cells and use the remaining, untouched mouse CD8+ T cells for any application
Description of Materials
Dynabeads are uniform, superparamagnetic polystyrene beads (4.5 μm diameter) coated with a polyclonal sheep anti-rat IgG antibody.
Additional Materials Required
Important Notes:
Dynabeads Washing Procedure
Dynabeads should be washed before use.
1. Resuspend the Dynabeads in the vial.
2. Transfer the desired volume of Dynabeads to a tube.
3. Add the same volume of Buffer 1, or at least 1 ml, and mix.
4. Place the tube in a magnet for 1 min and discard the supernatant.
5. Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Buffer 1 as the initial volume of Dynabeads.
Sample Preparation
See recommended procedure to prepare cells from mouse spleen or lymph nodes.
Critical Steps for Cell Isolation
Isolation of Mouse CD8+ T Cells fromSpleen or Lymph Node Leucocytes
This protocol is based on 1 x 107 leucocytes. It is scalable from 1 x 107-1 x 109 cells, (see table 1).
The supernatant contains the negatively isolated mouse CD8+ T cells.
Table 1. Volumes for mouse CD8+ T cell isolation per 1 x 107 starting leucocytes.
Working volume per 1 x 107 leucocytes | |
---|---|
Cell volume (step 1) | 100 μl |
FCS (step 2) | 20 μl |
Antibody Mix (step 3) | 20 μl |
Washing (step 5) | 2 ml |
Resuspension (step 6) | 800 μl |
Mouse Depletion Dynabeads (step 7) | 200 μl |
Volume added before magnet (step 10) | 1 ml |
Dynal MPC recommended | MPC-L/MPC-15/MPC-50 |
When working with higher cell numbers, scale up all reagents and volumes accordingly (e.g. for 2 x 107 leucocytes use twice the volume of all indicated reagent volumes). As a rule of thumb, up to 5 x 107 leucocytes can be processed in a single 15 ml tube and up to 2 x 108 leucocytes can be processed in a single 50 ml tube.
Downstream Applications
Isolated mouse CD8+ T cells can be used in applications such as cell culture, flow cytometry, functional assays and molecular studies.
Invitrogen Dynal AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.
Description of Materials
Dynabeads FlowComp™ are uniform, superparamagnetic beads (2.8 μm in diameter). Supplied at a concentration of approx. 1 × 109 beads (10 mg) per ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3) as preservatives.
Storage/Stability
This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.
Warnings And Limitations
This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.
Avoid pipetting by mouth!
Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .
For Research Use Only. Not for use in diagnostic procedures.