MojoSort™ anti-Biotin Nanobeads Column Protocol – Positive or Negative Selection

 

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 cocktail that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:2 to 1:10 for the antibody cocktail can be used. Dilutions ranging from 1:5 to 1:20 for the anti-Biotin 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™.

 

Positive vs. Negative Selection on columns: If your target cells are the labeled cells (the positive fraction), the desired cells are eluted from the column in step 5 of the “Example of magnetic separation with medium capacity columns” protocol below. If your target cells are the unlabeled cells (negative fraction), your desired cells are collected in step 3 and 4 of the “Example of magnetic separation with medium capacity columns” protocol below.

 

Protocol Steps

 

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 in MojoSort™ Buffer.
4. Aliquot 100 µL of cell suspension (107 cells) into a new tube. Check the recommended usage for flow cytometric staining of the Biotin-conjugated antibody indicated in the antibody technical datasheet. Calculate the volume to stain 107 cells (or desired amount of cells). Add the appropriate volume of pre-diluted Biotin-conjugated antibody to the cell suspension, mix well and incubate on ice for 15 minutes.
   Note: For the Biotin-conjugated antibodies, we recommend to do a titration to determine the optimal concentration.
5. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
6. Discard the supernatant and resuspend cells in 100 µL of MojoSort™ Buffer.
7. Resuspend the beads by vortexing, maximum speed, 5 touches. Add the appropriate volume of pre-diluted anti-Biotin Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, if the volume of pre-diluted Nanobeads for 1x10⁷ cells is 10 µL, add 100 µL for 1 x 10⁸ cells. When working with less than 10⁷ cells, use indicated volumes for 10⁷ cells.
   Note: The amount of Nanobeads to use always depends on the frequency of the target, among a few other factors. We recommend to do a titration to determine the optimal concentration.
8. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
9. Discard the supernatant.
10. Resuspend the cells in 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 in at least 500 µL of buffer to the column through a 30 µm filter and collect the fraction containing the unlabeled cells. For negative selection, these are the unlabeled cells of interest; do not discard.
4. Wash the cells in the column 2 times with 3 mL of buffer and collect the fraction containing the untouched cells. Combine with the collected fraction from step 3. For negative selection, these are the unlabeled cells of interest; do not discard.
5. Do not discard the column if you are performing positive selection. The labeled cells must be eluted from the column. Remove the column from the magnetic separator and it over a collection tube. Then add 5 mL of buffer and flush out the magnetically labeled fraction with a plunger or supplied device. For positive selection, these are the labeled 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.

 

 

Kit	Purity	Yield
Mouse CD19 Nanobeads	97.7%	94%

 

 

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.

B cells isolated with MojoSort™ CD19 nanobeads using separation columns.

B cells isolated with competitor's CD19 magnetic beads using separation columns.