MojoSort™ Human CD45 Selection Kit Column Protocol

Introduction

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 and/or antibodies when using columns provided by other commercial suppliers. Please consult the product webpage on the BioLegend website for any suggested dilutions based on in-house testing (under the “Product Details”, “Application Notes” section). However, we still recommend performing a titration to find the best dilution factor for your experiment and samples. Please contact BioLegend Technical Service (tech@biolegend.com) if further assistance is needed.

 

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 Steps

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

 

2. (If necessary) Make pre-diluted reagents as necessary for following separation steps.
   
    
3. 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.
   
    
4. 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.

 

5. Aliquot 100µL of cell suspension (10⁷ cells) for each sample into separate 5 mL (12 x 75 mm) polypropylene tubes. Add 10µL of the pre-diluted Human CD45 Positive Selection Cocktail. Mix well and incubate at room temperature for 5 minutes. Scale up the volume accordingly if separating more cells. For example, add 100µL of the pre-diluted Biotin-Antibody Cocktail for separating 1 x 10⁸ cells in 1mL 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.

 

6. Resuspend the beads by vortexing, maximum speed, 5 touches. Add 10µL of pre-diluted Streptavidin Nanobeads. Mix well and incubate at room temperature for 3 minutes. Scale up the volume accordingly if separating more cells. For example, add 100µL of pre-diluted 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.

 

7. Add the appropriate amount of MojoSort™ Buffer and proceed to separation. At least 500 µL is needed for column separation.
   Note: There are several types of commercially available columns, depending on your application. Choose the one that fits best your experiment:

 

 

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.
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 cells may be useful as controls, to monitor purity/yield, or other purposes.
5. 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. These are the positively isolated cells of interest; do not discard. To increase the purity of the magnetically labeled fraction repeat the isolation process with a new, freshly prepared column.