TITLE: MojoSort™ Selection Kits Protocol - 5
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Product description and procedure summary:</span></div><div class = "text-block">Target cells are either selected or depleted by incubating the sample with the biotin 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. If 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;">Sample Preparation:</span></div><div class = "text-block"><span>Enzymatic digestion of mouse brain followed by myelin removal is recommended to achieve the highest purity and yield. There are several protocols published that can be applied. As a general guideline, Trypsin digestion followed by a 70/37/30% percoll gradient will increase final purity and yield.</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></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 70µm 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 107 cells/mL
?. Aliquot 100µL of cell suspension (106 cells) into a new tube. Add 10µL of the Biotin-Antibody Cocktail. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, add 100 µL of Antibody Cocktail for separating 1 x 107 cells in 1 ml of MojoSort™ Buffer. When working with less than 106 cells, use indicated volumes for 106 cells.Optional: Take an aliquot before adding the cocktail to monitor purity and yield.
?. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes.
?. Discard supernatant and resuspend in 100µL of MojoSort™ Buffer.
?. Resuspend the beads by vortexing, maximum speed, 5 touches. Add 10µL of 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 Nanobeads for separating 1 x 107 cells in 1 ml of MojoSort™ Buffer. When working with less than 106 cells, use indicated volumes for 106 cells.
?. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes.
?. Discard supernatant.
?. Add 2.5mL 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 2.5mL MojoSort™ Buffer.
?. Repeat steps 10-12 on the labeled fraction twice more for a total of 3 separations. Pool the unlabeled fractions and keep the labeled cells. The fraction that is not of interest may be useful as staining controls, to monitor purity/yield, or other purposes.Optional: Take a small aliquot to monitor purity and yield.