MojoSort™ Human CD14 Selection 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 anti-human CD14,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.
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.
Without washing, add 10 μL ofStreptavidin 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.
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 10 and 11 with the magnetically labeledfraction 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.
Theyield will typically increase about 8 – 10% with a second separation, and about 2 – 5% with a thirdseparation.
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.
