MojoSort™ Whole Blood Human CD4 T Cell 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 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 less antibody cocktail that with other commercial suppliers. For recommended dilution factors based on internal testing, please consult the product webpage (noted under the “application notes” section). Some additional optimization may be required. 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™.

 

 

Note: Keep MojoSort™ Buffer on ice throughout the procedure.

           

1. Collect whole blood in collection tube that has anticoagulant, preferably EDTA
   1. Note: keep MojoSort Buffer on ice throughout the procedure.
2. Aliquot 1mL of freshly drawn anti-coagulated whole blood into a new tube. Add 20µL of the pre-diluted Biotin-Antibody Cocktail. Mix well and incubate at room temperature for 5 minutes. Scale up the volume if separating more cells. For example, add 40µL of pre-diluted antibody cocktail for separating 2mL blood. When working with less than 1mL blood, use indicated volumes for 1mL.
3. Vortex the Streptavidin conjugated Nanobeads (to resuspend) at max speed, 5 touches, and prepare the dilutions to test. Add 20µL Streptavidin Nanobeads. Mix well and incubate at room temperature for 5 minutes. Scale up the volume accordingly if separating more cells. For example, add 40µL of pre-diluted antibody cocktail for separating 2mL blood. When working with less than 1mL blood, use indicated volumes for 1mL.
4. 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:

 

	Max. volume of whole blood	Column rinse volume	Cell wash volume	Elution volume
Whole Blood Column	15 mL	3 mL	3x3 mL	5 mL

 

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 1 X Red Blood Cell Lysis Buffer (10X Cat. No. 420301 / 420302). Incubate 15min, in dark at room temperature followed by filtering through a cell strainer (40-70mm). Wash twice with Cell Staining Buffer (Cat. No. 420201) or equivalent..

 

If desired, the labeled cells can be collected by taking 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. The labeled cells may be useful as staining controls, to monitor purity/yield, or other purposes.