MojoSort™ Mouse CX3CR1 Selection Kit Protocol
Prepare cells from your tissue of interest.
Enzymatic digestion, followed by myelin removal, is strongly recommended.
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 40 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 107cells/mL.
Aliquot 100 μL of cell suspension (106 cells) into a new tube.
Add 10 μL of the Biotin‐CX3CR1 antibody, 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 107 cells.
When working with less than 106 cells, use indicated volumes for 106 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.
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 107 cells.
When working with less than 106 cells, use indicated volumes for 106 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.
Pour out and collect the liquid.
These are the cells of interest; DO NOT DISCARD.
If needed, add 3 mL of MojoSort™ Buffer and repeat steps 10 and 11 with the magnetically labeled fraction up to two times, and then pool the unlabeled fractions.
Notes: 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 beforeplacing the tube in the magnet to monitor purity and yield.
Scale up volumes accordingly if using a 14mL tube.
Place the tube in the magnet for 5 minutes.
