MojoSort™ Mouse NK 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 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.
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™.

Sample Preparation: Dead cell removal prior to cell isolation is recommended to achieve the highest purity and yield of mouse NK cells. Dead cells can be removed using appropriate centrifugation media or by other methods.
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 4mL 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 70µm 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 by adding MojoSort™ Buffer.
4. Aliquot 100µL (107 cells) into a new tube. Add 10µL 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 100µL of pre-diluted Antibody Cocktail for separating 1 x 108 cells in 1ml of MojoSort™ Buffer. When working with less than 107 cells, use indicated volumes for 107 cells.
5. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes.
6. Discard the supernatant and resuspend cells in 100uL of MojoSort™ Buffer.  
7. Vortex the Streptavidin conjugated Nanobeads (to resuspend) at max speed, 5 touches, and prepare the dilutions to test. Add 10µL 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.
8. Wash the cells by adding MojoSort™ Buffer up to 4mL. 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 500µL 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 in at least 500uL of buffer to the column through a 30µm filter and collect the fraction containing the unlabeled cells. These are the cells of interest; do not discard.
  4. Wash the cells in the column 2 times with 3mL of buffer and collect the fraction containing the untouched cells. Combine with the collected fraction from step 3.
  5. 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.
Data
Flow cytometry. High purity and yield. “After Isolation” plot shows purified population of interest using pre-diluted MojoSort™ reagents in separation columns.
KitPurityYield
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.
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