TITLE: MojoSort™ Whole Blood Human Neutrophil Isolation Kit Protocol
AUTHORS: Ken Lau

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol covers usage of BioLegend's MojoSort™ Human Neutrophil Isolation Kit Protocol.</div><div class = "text-block"><span>Please note that BioLegend is no longer updating protocols.io. For the most up-to-date protocols, visit our website: </span><a href="https://www.biolegend.com/en-us/technical-protocols" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.biolegend.com/en-us/technical-protocols</span></a></div></div>

[STEPS]
?. Collect whole blood in collection tube that has anticoagulant, preferably EDTA.Note: Keep MojoSort™ Buffer on ice throughout the procedure.
?. Aliquot 1 mL of human whole blood into a 5 mL (12 x 75 mm) polypropylene tube. Add 10 µL of the Biotin-Antibody Cocktail. Mix well and incubate on ice for 15 minutes. Scale up or down the volume accordingly if separating more blood. For example, add 20 µL of biotinylated antibody cocktail for separating 2 mL blood.
?. Resuspend the beads by vortexing, maximum speed, 5 touches. Add 10 µL of Streptavidin Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more volume of whole blood. For example, add 20 µL of Nanobeads for separating 2 mL of whole blood.
?. Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300 x g for 5 minutes. Remove supernatant by Pipet aid instead of pouring.
?. Add MojoSort™ Buffer, see Table 1 for volume. Note: If you observe aggregates, filter the suspension. To maximize yield, you can disrupt the aggregates by pipetting the solution up and down.Table 1. MojoSort™ Whole Blood Human CD8 Nanobeads Protocol: Separation Volumes and Magnet TimesCat. No.MojoSort™ Buffer VolumeMagnetic Incubation TimeMojoSort™ magnet480019For samples ≤ 1 mL:
Add 3mL buffer for separation5 minMojoSort™ 14 mL magnet480020For samples ranging 1 – 5 mL:
Top volume up to 10mL for each separation
(e.g. for 5 mL sample add 5 mL buffer for the first separation, then add 10 mL buffer for subsequent separations)10 min
Cat. No.MojoSort™ Buffer VolumeMagnetic Incubation TimeMojoSort™ magnet480019For samples ≤ 1 mL:
Add 3mL buffer for separation5 minMojoSort™ 14 mL magnet480020For samples ranging 1 – 5 mL:
Top volume up to 10mL for each separation
(e.g. for 5 mL sample add 5 mL buffer for the first separation, then add 10 mL buffer for subsequent separations)10 min
?. Place the tube in the magnet, see Table 1 for magnetic incubation time.
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 MojoSort™ Buffer.
?. Repeat steps 4-6 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.
?. Resuspend the magnetically labeled cells in desired volume, making sure to rinse the sides of the tube for better recovery.
?. (Optional) Lyse remaining erythrocytes using 20 mL room temperature 1X Red Blood Cell Lysis Buffer (10X Cat. No. 420301/420302). Incubate 15 min, in dark at room temperature followed by filtering through a cell strainer (40-70 µm). Wash twice with FACS wash buffer.
?. Chart Protocol: