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 Streptavidin Nanobeads can be used. Please contact BioLegend Technical Service (tech@biolegend.com) 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 (tech@biolegend.com) for more information and guidance. We do not recommend using MojoSort™ particles for BD’s IMag™ or Life Technologies’ DynaMag™.
1. 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.
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: For MojoSort™ Mouse CD4 Naïve T Cell Isolation Kit (480039, 480040), we recommend to use PBS without Ca/Mg in the final wash of your sample preparation, then resuspend the cells in PBS without Ca/Mg at an appropriate concentration (e.g. 1 x 108 cells/mL) and incubate on ice for 30 minutes.
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.
4. 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 cocktail 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.
5. 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.
6. 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:
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 CD4 T Cell Isolation Kit | 95.7% | 85% |
Electron Microscopy. CD4+ T cells Isolated with MojoSort™ CD4 T Cell Isolation Kit using columns do not display particles in the cell surface. Image is representative of 36 different cells
CD4+ T cells isolated with MojoSort™ CD4 T Cell Isolation Kit using separation columns.