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 23 and 24 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 .
