TITLE: MojoSort™ Streptavidin Nanobeads Column Protocol - Negative Selection
AUTHORS: Kelsey Miller, Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with </span><span style = "font-weight:bold;">pre-diluted </span><span>MojoSort™ reagents and using the columns as indicated by the manufacturer.</span></div><div class = "text-block"><span style = "font-weight:bold;">Note: </span><span>Due to the properties of our beads, it may be possible to use far fewer beads and less antibody cocktail that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:2 to 1:10 for the antibody cocktail can be used. Dilutions ranging from 1:5 to 1:20 for the Streptavidin Nanobeads can be used. Please contact BioLegend Technical Service (tech@biolegend.com) if further assistance is needed.</span></div></div>

[STEPS]
?. Prepare cells from your tissue of interest or blood 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) polypropylene tube.Note: Keep MojoSort™ Buffer on ice throughout the procedure.
?. Filter the cells with a 70 µm cell strainer, centrifuge at 300xg for 5 minutes, and resuspend in an appropriate volume of MojoSort™ Buffer. Count and adjust the cell concentration to 1 x 108 cells/mL by adding MojoSort™ Buffer.
?. Aliquot 100 µL of cell suspension (107 cells) into a new tube. Check the recommended usage for flow cytometric staining of the Biotin-conjugated antibody indicated in the antibody technical datasheet. Calculate the volume to stain 107 cells (or desired amount of cells). Add the appropriate volume of pre-diluted Biotin-conjugated antibody to the cell suspension, mix well and incubate on ice for 15 minutes.Note: For the Biotin-conjugated antibodies, we recommend to do a titration to determine the optimal concentration.
?. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
?. Discard the supernatant and resuspend cells in 100 µL of MojoSort™ Buffer.
?. Resuspend the beads by vortexing, maximum speed, 5 touches. Add the appropriate volume 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, if the volume of pre-diluted Nanobeads for 1x107 cells is 10 µL, add 100 µL for 1 x 108 cells. When working with less than 107 cells, use indicated volumes for 107 cells.Note: The amount of Nanobeads to use always depends on the frequency of the target, among a few other factors. We recommend to do a titration to determine the optimal concentration.
?. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
?. Discard the supernatant.
?. Resuspend the cells in appropriate amount of MojoSort™ Buffer and proceed to separation. 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:
?. [Example of magnetic separation with medium capacity columns: ]
Place the column in a magnetic separator that fits the column.
?. [Example of magnetic separation with medium capacity columns: ]
Rinse the column with 3 mL of cell separation buffer.
?. [Example of magnetic separation with medium capacity columns: ]
Add the labeled cell suspension in at least 500 µL of buffer to the column through a 30 µm filter and collect the fraction containing the unlabeled cells. These are the cells of interest; do not discard.
?. [Example of magnetic separation with medium capacity columns: ]
Wash the cells in the column 2 times with 3 mL of buffer and collect the fraction containing the untouched cells. Combine with the collected fraction from step 3.
?. [Example of magnetic separation with medium capacity columns: ]
If desired, the labeled cells can be collected by taking away the column from the magnet and place it on a tube. Then add 5 mL of buffer and flush out the magnetically labeled fraction with a plunger or supplied device. The labeled cells may be useful as staining controls, to monitor purity/yield, or other purposes.