Mojosort™ Mouse Neutrophil Isolation Kit Protocol
Pour out and collect the liquid .
These are the cells of interest ; DO NOT DISCARD .
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 filling up to 4 mL 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 an appropriate 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 .
Wash the cells by adding MojoSort™ Buffer up to 4 mL ; centrifuge the cells at 300 x g for 5 minutes .
Discard supernatant and resuspend in 100 μL of MojoSort™ Buffer , or volume needed to keep the cells at 1 x 108 cells / mL .
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 volumes for 107 cells .
Add MojoSort™ Buffer up to 2 mL and place the tube in the magnet for 5 minutes place the tube directly in the magnet for 5 minutes .
Collect the liquid in a clean tube .
DO NOT DISCARD .
Optional : Take an aliquot before placing the tube in the magnet to monitor purity and yield .
Remove the tube from the magnet and resuspend the cells in 2 mL of MojoSort™ Buffer .
Place it back into the magnet for 5 minutes .
Collect the liquid in the same tube as in step 8 .
DO NOT DISCARD .
Note : If you observe aggregates , filter the suspension .
To maximize yield , you can disrupt the aggregates by pipetting the solution up and down .
Take the pooled negative fraction tube ( should contain 4 mL in addition to the sample volume and Nanobeads volume ) and place the tube in the magnet for 5 minutes .
