MojoSort™ Human CD4 T Cell Selection 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 5 μL of the biotin anti-human CD14 antibody, mix well and incubate on ice for 15 minutes.
Scale up the volume accordingly if separatingmore cells.
For example, add 50 μL for 1 x 108 cells.
When working with less than 107 cells, use indicatedvolumes for 107 cells.
Optional: Take an aliquot before adding the antibody to monitor purity and yield.
Resuspend the Streptavidin Nanobeads by vortexing, maximum speed, 5 touches.
Without washing, 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.
Resuspend the cells in 3 mL of MojoSort™ Buffer.
Place the tube in the magnet for 5 minutes.
Collect the liquid in a new tube.
This fraction contains the CD4+ T Cells; DO NOT DISCARD.
Centrifuge at 300 x g for 5 minutes, discard supernatant.
Resuspend by flicking or in 100 uL of MojoSort™ buffer.
Resuspend the CD4 Nanobeads 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.10.
Add 3 mL of MojoSort™ Buffer.
Place the tube in the magnet for 5 minutes.
Pour out the liquid.
Recover the tube and resuspend the cells in appropriate amount of buffer.
These are the CD4+ T Cells.
Repeat steps 10 – 12 on the labeled fraction 2 more times, for a total of 3 magnetic separations.
Optional: Take a small aliquot to monitor purity and yield.
