MojoSort™ Mouse NK Cell Isolation Kit Protocol
Prepare cells from your tissue of interest without lysing erythrocytes.
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) polystyrene tube.
Note: Keep MojoSort™ Buffer on ice throughout the procedure.
Filter the cells with a 70 μm cell strainer, centrifuge at 300 x g for 5 minutes, and resuspend in anappropriate volume of MojoSort™ Buffer.
Count and adjust the cell concentration to 1 x 108 cells/mL.
Aliquot 100 μL of cell suspension (107 cells) into a new tube.
Add 10 μL of the Biotin-Antibody Cocktail, mix well and incubate on ice for 15 minutes.
Scale up the volume accordingly if separating more cells.
For example, add 100 μL for 1 x 108 cells.
When working with less than 107 cells, use indicated volumes for 107 cells.
Optional: Keep unused cells, or take an aliquot before adding the cocktail to monitor purity and yield.
Resuspend the beads by vortexing, maximum speed, 5 touches.
Add 10 μL of 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 for 1 x 108 cells.
When working with less than 107 cells, use indicated volumesfor 107 cells.
Wash the cells by adding 3 mL of MojoSort™ Buffer; centrifuge at 300 x g for 5 minutes, discard supernatant.
Optional: Take an aliquot before placing the tube in the magnet to monitor purity and yield.
Resuspend the cells in 3 mL of MojoSort™ Buffer.
Note: If you observe aggregates, filter the suspension.
To maximize yield, you can disrupt the aggregates by pipetting the solution up and down.
Place the tube in the magnet for 5 minutes.
Pour out and collect the liquid.
These are your cells of interest; DO NOT DISCARD.
If needed, add 3 mL of MojoSort™ Buffer and repeat steps 8 and 9 with the magnetically labeled fraction up to two times, and then pool the unlabeled fractions.
Note: Repeating the magnetic separation increases the yield, without a strong impact on the purity.
The yield will typically increase about 8 – 10% with a second separation, and about 2 – 5% with a third separation.
The purity may decrease 1 – 2% with each separation.
Optional: Take a small aliquot before placing the tube in the magnet to monitor purity and yield.
