Dynabeads FlowComp™ Flexi, part A – flow compatible and tube-based cell isolation

This protocol describes magnetic labeling and isolation of cells from 5 X 10⁷ cells using Dynabeads FlowComp Flexi.  When working with fewer cells than 5 X 10⁷ use the same volumes as indicated. When working with higher cell numbers,  scale up all reagent volumes and total volumes accordingly.

Preparations

• DSB-X label your antibodies following the procedure recommended in the DSB-X Biotin Protein Labeling Kit (or use previously DSB-X labeled antibodies)
• Prepare a single cell suspension of 1 X 10⁸ cells/ml in Isolation Buffer
• Prepare approximately 10 ml of Isolation Buffer per 5 X 10⁷ cells

Isolation Procedure

1. Prepare antibody: The most optimal antibody concentration should be determined for a new cell isolation. We recommend using 5-50 µg mAb/5  x 10⁷ cells. Adjust the concentration of the labeled antibody accordingly using PBS w/ 5% BSA (optional: 0.2% Sodium Azide).
2. Resuspend 5 X 10⁷ cells in 500 µl Isolation Buffer and add 25 µl DSB-X labeled antibody.
3. Mix well and incubate for 10 min. at 2-8° C.
4. Add 2 ml cold Isolation Buffer to wash the cells, followed by centrifugation for 8 min. at 350 X g.
5. Remove and disregard the supernatant.
6. Add 1 ml cold Isolation buffer to the cell pellet and resuspend by pipetting
7. Add 75 µl resuspended FlowComp Dynabeads and mix well. (e.g. vortex for 2-3 seconds). Increased volume of Dynabeads may increase recovery.
8. Incubate for 15 min at 2-8°C with mixing providing constant tilting and rotation.
9. Place the tube in the magnet for minimum of 1 min. Carefully remove and discard the supernatant, while the tube is still in the magnet.
10. Remove the tube from the magnet. Add at least 1 ml cold Isolation Buffer and resuspend the bead-bound cells by gently pipetting 5 times.
11. Place the tube in the magnet for a minimum for 1 min, Carefully remove and discard the supernatant
12. Remove the tube from the magnet and carefully resuspend the bead-bound cells in 1 ml FlowComp Release Bufffer
13. Incubate for 10 min. at room  temperature with rolling and tilting.
14. Pipette 10 times to efficiently release the cells and place the tube in the magnet for 1 min.  Avoid air bubbles.
15. Carefully transfer the supernatant containing the beads-free cells to a new tube and place the tube in the magnet for 1 min to remove all residual beads.
16. Transfer the supernatant containing the bead-free cells to the new tube.

The cells can be used directly for flow staining. For cell culture; centrifuge and resuspend in cell media of choice.

Invitrogen Dynal AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

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.

Choice of antibody
The choice of antibody clone is the most important factor for successful cell isolation. Please note that not all antibodies are suitable for cell isolation with magnetic beads, although proven successful for staining.

Antibody-coating of cells

• Very low levels of target cells may require larger amounts of antibody, longer incubation time or higher cell concentration. In general, the concentration of antibody during coating of cells is a very important factor for good results.
• Coating temperature can be varied in the range of 0-37°C. 2-8°C is usually preferred to reduce biological activity in the cell (e.g. enzymatic cleavage or internalization of receptors) while keeping coating time as short as possible.
• Coating time can be optimized in the range of 5-30 minutes. 10 minutes is usually sufficient. Coating on ice  (compared to 2–8°C) will require longer coating time (e.g. 20 minutes).

Storage and handling of conjugates
Please follow the recommendations in the DSB-X Biotin Protein Labeling Kit. When stored correctly at 4°C, the conjugate should be stable for several months.

Cell isolation and release

• Cell isolation time can be optimized in the range of 5-30 minutes. 10-20 minutes is usually sufficient for optimal recovery.
• If the target cell concentration exceeds 50% of the total cells, reduce the cell concentration accordingly. Alternatively, increase the amount of beads above 75 μl/ml of cell sample. For isolation of rare cells (e.g. stem cells from BM or leukophoresis) the cell concentration should be increased up to 1 × 10⁸ cells/ml.
• The temperature applied for cell isolation and release may affect the efficacy of the system. Cell isolation and release can be performed in the range of 0-37°C. For isolation of phagocytic cells (i.e. monocytes or macrophages), keep the temperature low to avoid phagocytic activity.
• The release time can be optimized in the range of 2–20 minutes. 2–10 minutes is usually sufficient. Another release step can also be added if necessary.
• Increase the number of washing steps if necessary.