MojoSort™ Whole Blood Human Neutrophil Isolation 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 that 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. 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™.

 

Protocol Steps

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1. Collect whole blood in collection tube that has anticoagulant, preferably EDTA.
   Note: Keep MojoSort™ Buffer on ice throughout the procedure.


2. Aliquot 1mL of human whole blood into a 5 mL (12 x 75 mm) polypropylene tube. Add 5µL of the Biotin-Antibody Cocktail. Mix well and incubate on ice for 15 minutes. Scale up or down the volume accordingly if separating more blood. For example, add 10 µL of biotinylated antibody cocktail for separating 2mL blood.


3. 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 larger starting volume of whole blood. For example, add 20 µL of pre-diluted Nanobeads for separating 2mL of whole blood.


4. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes. Remove supernatant by Pipet aid instead of pouring.


5. Proceed to separation on column as indicated by the manufacturer.
   Note: There are several types of commercially available columns, depending on your application. Choose the one that fits best your experiment. Note: This kit requires the use of a column compatible with processing of human whole blood.

 

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Columns:

 

Example of magnetic separation with whole blood 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 whole blood 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 elution 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.
6. (Optional) Lyse remaining erythrocytes using 20mL room temperature 1XRed Blood Cell Lysis Buffer (10X Cat. No. 420301 / 420302). Incubate 15min, in dark at room temperature followed by filtering through a cell strainer (40-70m). Wash twice with FACS wash buffer.