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 .
