MojoSort™ Negative Selection Columns Protocol
Prepare a single cell suspension and resuspend the cells with ice cold cell separation buffer (MojoSort™buffer recommended).
Pass the cells through a 70 μm filter, centrifuge (300 x g for 5 minutes), discard the supernatant and resuspend the cells in cell separation buffer.
Adjust the cell concentration to 1 x 108 cells/mL.
Aliquot 100 μL (107 cells) into a new tube.
Add 10 μL of the pre-diluted Biotin-Antibody Cocktail, mix well and incubate on ice for 15 minutes.
Scale up the volume if separating more cells.
For example, add 100 μL of pre-diluted antibody cocktail for separating 1 x 108 cells in 1ml of buffer.
When working withless than 107 cells, use indicated volumes for 107 cells.
Add cell separation buffer up to 4 mL; centrifuge the cells at 300 x g for 5 minutes.
Discard supernatant and resuspend in 100 μL of buffer.
Vortex the Streptavidin conjugated Nanobeads (to resuspend) at max speed, 5 touches, and prepare the dilutions to test.
Add 10 μL of pre-diluted 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 of pre-diluted Nanobeads for separating 1 x 108 cells in 1ml of buffer.
When working with less than 107 cells,use indicated volumes for 107 cells.
Note: Depending on the isolation kit you are using, a wash step may be required here.
If you observe aggregates, filter the suspension.
To maximize yield, you can disrupt the aggregates by pipetting the solution up and down.
Resuspend the cells in appropriate amount of buffer.
At least 500 μL is needed for column separation, Note: There are several types of commercially available columns, depending on your application, choose the one that fits best your experiment.
See 'Guidelines' for choosing a column for your experiment.
