MojoSort™ Mouse CD117 (c-Kit) 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 less antibody 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. 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. 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 a small volume of MojoSort™ Buffer. Count and adjust the cell concentration to 1 x 10⁸ cells/mL by adding MojoSort™ Buffer.
4. Aliquot 100 µL of cell suspension (10⁷ cells) into a new tube.
5. Add 5 µL of TruStain FcX™ (anti-mouse CD16/32) Antibody. Mix well and incubate at room temperature for 10 minutes. Scale up the volume accordingly if separating more cells. For example, if the volume of TruStain FcX™ for 1x10⁷ cells is 5 µL, add 50 µL for 1 x 10⁸ cells in 1 mL of MojoSort™ Buffer. When working with less than 10⁷ cells, use indicated volumes for 10⁷ cells.
6. Add 10 µL of the pre-diluted PE anti-mouse CD117 (c-Kit) antibody. Mix well and incubate on ice for 15 minutes. Scale up the volume if separating more cells. For example, add 100 µL of pre-diluted Antibody 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. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
8. Discard the supernatant and resuspend cells in 100 uL of MojoSort™ Buffer.
9. Resuspend the beads by vortexing, maximum speed, 5 touches. Add 10 µL of pre-diluted Mouse anti-PE Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, add 100 µL 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.
10. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
11. Discard the supernatant.
12. 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 3mL of cell separation buffer.
3. Add the labeled cell suspension to the column through a 30µm filter and collect the fraction containing the unlabeled cells.
4. Wash the cells in the column 3 times with 3mL 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 5mL 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.

Data

Flow cytometry. High purity and yield. "After Isolation" plot shows purified population of interest using pre-diluted MojoSort™ reagents in separation columns.

 

Electron Microscopy. MojoSort™ Nanobead-isolated CD19⁺cells using columns do not display more bound beads on the cell surface (A) as compared to cells isolated with a compatible commercial product using the same columns (B). Red arrows indicate where the particles are located. Numbers indicate either 2 or 3 magnetic particles adjacent to each other. Pictures were taken at the same magnification, scale shown in B. Images are representative of 41 different cells each.