MojoSort™ Mouse Neutrophil 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 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.
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.
Pour out and collect the liquid.
These are the cells of interest; DO NOT DISCARD.
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.
