MojoSort™ Dead Cell Removal Column Protocol

Introduction

This protocol is applicable to the use of both MojoSort™ Mouse Dead Cell Removal and MojoSort™ Human Dead Cell Removal kits on commercially available magnetic columns available from other vendors. BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.
 

Note: Due to the properties of our beads, it may be possible to use far fewer beads than with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used.

 

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™.

 

Protocol Steps

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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
   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 pre-diluted Apo-Monomer and 10µL of pre-diluted 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) into separate tubes for each sample. Add 20µL of the monomer-streptavidin complex (from step 4) into each tube. 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.
    
7. Add the appropriate amount of MojoSort™ Buffer and proceed to separation. At least 500 µL is needed for column separation.

 

Columns:

 

Example of magnetic separation with medium capacity columns:

1. Place the column in a magnetic separator that fits the column.
2. Rinse the column with 3 mL of cell separation buffer.
3. Add the labeled cell suspension to the column through a 30 µm filter and collect the fraction containing the unlabeled cells. These are the live cells.
4. Wash the cells in the column 3 times with 3 mL of buffer and collect the fraction containing the unlabeled cells. Combine with the collected fraction from step 3. These are also live cells.
5. (optional) Take away the column from the magnet and place it on a tube. Then add 5 mL of buffer and flush out the magnetically labeled fraction with a plunger or supplied device. This fraction contains the dead cells – these cells may be useful as controls to monitor purity/yield, or other purposes.