MojoSort™ Dead Cell Removal Protocol

Introduction

This protocol is applicable to the use of both MojoSort™ Mouse Dead Cell Removal and MojoSort™ Human Dead Cell Removal kits on the MojoSort™ magnets (Cat. No. 480019/480020) or equivalent. Please carefully read through the entire protocol before starting the experiment.

 

Reagent List

MojoSort™ (Human or Mouse) Dead Cell Removal Kit 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 Tube for monomer-streptavidin nanobead complex formation (e.g. 1.5 mL microfuge tube) 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™. A separate protocol for the use of this kit on column-based magnetic separators is available (list of protocols adapted for column use available here: biolegend.com/mojosort/columnIndex).

 

Protocol

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Product description and procedure summary: Target cells are depleted by incubating the sample with preassembled biotin recombinant protein and magnetic Streptavidin Nanobeads (Cat. No. 480015/480016) mix. 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 1x10⁷ cells per tube. See additional notes on each step for further instructions if using fewer cells. Some optimization may be required to determine optimal conditions for 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.

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1. Prepare cells from your tissue of interest or blood without lysing erythrocytes. Kits for human samples have been optimized for PBMCs, please prepare the cells using a suitable method.
    
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 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.
   1. If working with fewer than 1x10⁷ cells, resuspend in 100µL sample volume
       
4. Preassemble the monomer-streptavidin complex:
   1. For each sample, add 10µL of Apo-Monomer and 10µL of Streptavidin Nanobeads in the same tube. Mix well by pipetting up and down several times. Scale up volumes for each component respectively if separating multiple samples.
   2. Incubate at room temperature for 5 minutes then proceed to next step.
       
5. Aliquot 100µL of cell suspension (1x10⁷ cells, or fewer) for each sample into separate 5 mL (12 x 75 mm) polypropylene tubes. Add 20µL of the monomer-streptavidin complex (from step 4) into each sample. Mix well and incubate on ice for 15 minutes.
   1. Scale reagent use to cell count accordingly. We observed higher purity and yield from samples containing fewer cells (~1x10⁶ cells, from cryopreserved PBMCs) with adding 8x less monomer-streptavidin complex (2.5µL). Some optimization may be required.
       
6. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes. Remove and discard the supernatant carefully without disrupting the cells pelleted.
    
7. 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.
    
8. Place the tube in the magnet for 5 minutes.
    
9. Pour out and collect the liquid. These are your cells of interest; DO NOT DISCARD.
    
10. Repeat steps 7-9 with labeled cells once more for a total of 2 separations. Pool the unlabeled fractions. The labeled cells may be useful as staining controls, to monitor purity/yield, or other purposes.
    
    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.

 

Schematic 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 for further assistance on how to use MojoSort™ Nanobeads in magnetic separation columns.