MojoSort™ Selection Kits Column Protocol - 3
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 (firstname.lastname@example.org) if further assistance is needed.
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 (email@example.com) for more information and guidance. We do not recommend using MojoSort™ particles for BD’s IMag™ or Life Technologies’ DynaMag™.
- Prepare cells from your tissue of interest or blood without lysing erythrocytes. Kits for human samples have been optimized for PBMCs, please prepare the cells using a suitable method.
- 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.
- 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.
- Aliquot 100 µL (107 cells) into a new tube. Add 10 µL of the pre-diluted Biotin-Antibody Cocktail. 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.
- Vortex the Streptavidin conjugated Nanobeads (to resuspend) at max speed, 5 touches, and prepare the dilutions to test. Add 10 µL of pre-diluted Streptavidin 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.
- Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
- Discard the supernatant.
- 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:
Example of magnetic separation with medium capacity columns:
- Place the column in a magnetic separator that fits the column.
- Rinse the column with 3 mL of cell separation buffer.
- Add the labeled cell suspension to the column through a 30 µm filter and collect the fraction containing the unlabeled cells.
- 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.
- 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.
Flow cytometry. High purity and yield. “After Isolation” plot shows purified population of interest using pre-diluted MojoSort™ reagents in separation columns.
|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.