MojoSort™ anti-Biotin Nanobeads Protocol - Positive Selection

 

Reagent List

MojoSort™ Buffer (5X) (Cat. No. 480017) MojoSort™ Magnet (Cat. No. 480019/480020) or compatible magnetic separation system Adjustable pipettes 70 µm filters (one per sample)

5 mL (12 x 75mm) or 14 mL (17 x 100mm) polypropylene tubes polypropylene tubes Reagents for sample preparation Reagents and instruments (Flow cytometer) to determine yield and purity

Important Note

MojoSort™ magnetic particles can be used with other commercially available magnetic separators, both free standing magnets and column-based systems. Because MojoSort™ protocols are optimized for the MojoSort™ separator, the protocols may need to be adjusted for other systems. Please contact BioLegend Technical Service (tech@biolegend.com) for more information and guidance. We do not recommend using MojoSort™ particles for BD’s IMag™ or Life Technologies’ DynaMag™.

 

Protocol Steps

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Product description and procedure summary:

 

If your target cells are the labeled cells (the positive fraction), use the MojoSort™ anti-Biotin Nanobeads Protocol – Positive Selection. If your target cells are the unlabeled cells (negative fraction), use the MojoSort™ anti-Biotin Nanobeads Protocol - Negative Selection.

 

Target cells are isolated by incubating your sample with a Biotin-conjugated antibody or antibody cocktail, followed by incubation with magnetic MojoSort™ anti-Biotin Nanobeads (Cat. No. 480032/480034). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells in the supernatant are discarded (or collected for control samples). The cells which are bead-bound and retained by the magnet are the cells of interest. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.

Note: This procedure is optimized for the isolation of 10⁷ to 2 x 10⁸ cells per tube. If working with fewer than 10⁷ cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. Scale up volumes if using 14 mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

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1. Prepare cells from your tissue of interest or blood without lysing erythrocytes.
2. In the final wash of your sample preparation, resuspend the cells in MojoSort™ Buffer by adding up to 4 mL in a 5 mL (12 x 75 mm) polypropylene tube.
   Note: Keep MojoSort™ Buffer on ice throughout the procedure.
3. Filter the cells with a 70 µm cell strainer, centrifuge at 300xg for 5 minutes, and resuspend in an appropriate volume of MojoSort™ Buffer. Count and adjust the cell concentration to 1 x 10⁸ cells/mL.
4. Aliquot 100 µL of cell suspension (10⁷ cells) into a new tube. Check the recommended usage for flow cytometric staining of the Biotin-conjugated antibody indicated in the antibody technical datasheet. Calculate the volume to stain 10⁷ cells (or desired amount of cells). Add the appropriate volume (see General Tips and FAQ below) of Biotin-conjugated antibody to the cell suspension, mix well and incubate on ice for 15 minutes.
   Optional: Take an aliquot before adding the cocktail to monitor purity and yield.
5. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
6. Discard supernatant and resuspend in 100 µL of MojoSort™ Buffer.
7. Resuspend the beads by vortexing, maximum speed, 5 touches. Add the appropriate volume (see General Tips and FAQ below) of Anti-biotin Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, if the volume of Nanobeads for 1x10⁷ cells is 10µL, add 100µL for 1 x 10⁸ cells. When working with less than 10⁷ cells, use indicated volumes for 10⁷ cells.
8. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes.
9. Discard the supernatant.
10. Add 2.5mL of MojoSort™ Buffer.
    Note: If you observe aggregates, filter the suspension. To maximize yield, you can disrupt the aggregates by pipetting the solution up and down.
11. Place the tube in the magnet for 5 minutes.
    Optional: Take a small aliquot before placing the tube in the magnet to monitor purity and yield. Keep unused cells to be used as control or other applications if needed.
12. Pour out the unlabeled fraction but you may want to keep the negative fraction for controls. Resuspend the labeled cells in 2.5 mL MojoSort™ Buffer.
13. Repeat steps 10-12 on the labeled bead-bound fraction once more for a total of 3 separations.
    Note: Repeating the magnetic separation increases the yield, without a strong impact on the purity. The yield will typically increase about 8-10% with a second separation. The purity may decrease 1-2% with each separation.

Chart Protocol:

 

Application notes: To use this product in magnetic separation columns, a titration of the Nanobeads should be performed. Optimal concentration for magnetic separation columns is lot-specific. Please contact BioLegend Technical Service (tech@biolegend.com) for further assistance on how to use MojoSort™ Nanobeads in magnetic separation columns.

 

General Tips & FAQ

 

• How much of Biotinylated antibodies should I use?
  • For the biotinylated antibodies, we recommend to do a titration to determine the optimal concentration. In general, if using only a single biotin-conjugated antibody (and not a cocktail), then using the flow cytometry recommended concentration may work okay as long as you are washing after antibody incubation. If you are using a cocktail or plan to remove the wash after antibody incubation, you should perform titrations and optimizations.
• How much of Anti-Biotin Nanobeads should I use?
  • The amount of beads to use always depends on the frequency of the target, among a few other factors. The standard volume to use is 10 µL of beads per 10⁷ cells. You can benefit from titrating the beads, using 2, 5, 10, and 20 µL of beads (for example). In some cases less than 10 µL of beads is enough to achieve the purity and you can save on the reagent. Please note that anti-Biotin Nanobeads volume should not exceed more than 20% of the total volume.

 

Note: The Antibody/Nanobead volume to add should not exceed more than 20% of the 100 µL cell suspension volume. Thus, for 100 µL of cell suspension do not add more than 20 µL of Antibody/Nanobeads. If you need to add more than 20µL, resuspend the cells in step 3 or step 6 at a higher concentration. For example, to add 50µL of Antibody/Nanobeads, resuspend the cells to a final concentration of 2 x 10⁸ cells/mL. You can then add 50 µL of Antibody/Nanobeads to 50 µL of cells. Always keep the total volume around 100 µL.