MojoSort™ Human CD45 Nanobeads Protocol B
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 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.
Resuspend the beads by vortexing, maximum speed, 5 touches.
Add 10 μL of 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 4 mL and centrifuge the cells at 300 x g for 5 minutes.
Resuspend the cells in 3 mL of MojoSort™ Buffer.
Optional: Take an aliquot before placing the tube in the magnet to monitor purity and yield.
Place the tube in the magnet for 5 minutes.
Pour out the liquid containing the unlabeled fraction.
Remove the tube from the magnet and resuspend the first labeled fraction in appropriate amount of buffer.
Place the tube containing the unlabeled fraction back in the magnet for 5 minutes.
Pour out the liquid containing the unlabeled fraction from the second magnetic incubation.
These are the CD45- cells, ready to use as needed.
Remove the tube from the magnet and use the fraction obtained in step 10 to resuspend this second labeled fraction and pool them together.
These are the CD45+ cells, ready to use as needed.
Optional: Take a small aliquot to monitor purity and yield.
If desired, pool the unlabeled fractions and process simultaneously with the positive labeled cells when assessing purity and yield.
