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.
