TITLE: MojoSort™ Streptavidin Nanobeads Protocol - Negative Selection
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Product description and procedure summary:</span><span style = "font-weight:bold;">
</span></div><div class = "text-block"><span style = "font-weight:bold;">If your target cells are the labeled cells (the positive fraction), use the Streptavidin Nanobeads Protocol – Positive Selection. If your target cells are the unlabeled cells (negative fraction), use the Streptavidin Nanobeads Protocol - Negative Selection.</span><span>
</span></div><div class = "text-block">Target cells are isolated by incubating your sample with a Biotin-conjugated antibody or antibody cocktail, followed by incubation with magnetic Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.
</div><div class = "text-block"><span style = "font-weight:bold;">Note:</span><span> This procedure is optimized for the isolation of 10</span><span style = "vertical-align:super;">7 </span><span>to 2 x 10</span><span style = "vertical-align:super;">8 </span><span>cells per tube. If working with fewer than 10</span><span style = "vertical-align:super;">7 </span><span>cells, keep volumes as indicated for 10</span><span style = "vertical-align:super;">7 </span><span>cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. </span><span style = "font-style:italic;">Scale up volumes if using 14 mL tubes and Magnet, and place the tube in the magnet for 10 minutes.</span><span></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.
?. Aliquot 100µL 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 (see General Tips and FAQ below) of Biotin-conjugated antibody to the cell suspension, mix well and incubate on ice for 15 minutes.Optional: Take an aliquot before adding the cocktail to monitor purity and yield.
?. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
?. Discard supernatant and resuspend in 100 µL of MojoSort™ Buffer.
?. Resuspend the beads by vortexing, maximum speed, 5 touches.Add the appropriate volume (see General Tips and FAQ below) of 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 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.
?. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
?. Discard the supernatant.
?. Add 2.5 mL of MojoSort™ Buffer.Note: If you observe aggregates, filter the suspension. To maximize yield, you can disrupt the aggregates by pipetting the solution up and down.
?. Place the tube in the magnet for 5 minutes.Optional: Take a small aliquot before placing the tube in the magnet to monitor purity and yield. Keep unused cells to be used as control or other applications if needed.
?. Pour out the unlabeled fraction. If these are your cells of interest, DO NOT DISCARD. Resuspend the labeled cells in 2.5 mL MojoSort™ Buffer.
?. Repeat steps 10-12 on the labeled fraction once more for a total of 2 separations. Pool the unlabeled fractions. The labeled cells may be useful as staining controls, to monitor purity/yield, or other purposes.Note: Repeating the magnetic separation increases the yield, without a strong impact on the purity. The yield will typically increase about 8-10% with a second separation. The purity may decrease 1-2% with each separation.