MojoSort™ Mouse Neutrophil Isolation Kit Protocol

 

Reagent List

  • MojoSort™ Buffer (5X) (Cat. No. 480017)
  • MojoSort™ Magnet (Cat. No. 480019/480020) or compatible magnetic separation system
  • Adjustable pipettes
  • 70µm filters (one per sample)
  • 5mL (12 x 75mm) or 14mL (17 x 100mm) 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

Product description and procedure summary:

Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. 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 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

 

  1. Prepare cells from your tissue of interest or blood without lysing erythrocytes.  

  2. In the final wash of your sample preparation, use PBS without Ca/Mg, then resuspend the cells in PBS without Ca/Mg at an appropriate concentration (e.g. 1 x 108 cells/mL) and incubate on ice for 30 minutes.
    Note: Keep MojoSort™ Buffer on ice throughout the procedure.  

  3. Filter the cells with a 70µm 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 100µL of cell suspension (107 cells) into a new tube. Add 10µL of the Biotin-Antibody Cocktail. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, add 100 µL of Antibody for separating 1 x 108 cells in 1 ml of MojoSort™ Buffer. When working with less than 107 cells, use indicated volumes for 107 cells.
    Optional: Take an aliquot before adding the cocktail to monitor purity and yield.

  5. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes.

  6. Discard supernatant and resuspend in 100µL of MojoSort™ Buffer.  

  7. Resuspend the beads by vortexing, maximum speed, 5 touches. Add 10µL of Streptavidin Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, add 100 µL of Nanobeads for separating 1 x 108 cells in 1 ml of MojoSort™ Buffer. 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 supernatant.  

  10. Add 2mL 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, DO NOT DISCARD. Resuspend the labeled cells in 2mL MojoSort™ Buffer.  

  13. Place the tube in the magnet for 5 minutes.  

  14. Pour out the unlabeled fraction and pool with the previously collected unlabeled cells (should contain ~4mL buffer and cells).  

  15. Place the pooled unlabeled fraction in the magnet for 5 minutes.

  16. Pour out the unlabeled fraction, these are the cells of interest, DO NOT DISCARD. The labeled fraction may be useful as staining controls, to monitor purity/yield, or other purposes.
    Optional: Take a small aliquot to monitor purity and yield.  
     

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.