TITLE: MojoSort™ Human anti-PE Nanobeads Column Protocol
AUTHORS: 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 that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the 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. Add 5 µL of Human TruStain FcX™ (Fc Receptor Blocking Solution), mix well and incubate at room temperature for 10 minutes. Scale up the volume accordingly if separating more cells. For example, add 50 µL of Human TruStain FcX™ for separating 1 x 108 cells in 1 ml of MojoSort™ Buffer. When working with less than 107 cells, use indicated volumes for 107 cells.
?. Check the recommended usage for flow cytometric staining of the PE-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 PE-conjugated antibody to the cell suspension, mix well and incubate on ice for 15 minutes.Note: For the PE-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 100uL of MojoSort™ Buffer.
?. Vortex the anti-PE Nanobeads (to resuspend) at max speed, 5 touches, and prepare the dilutions to test. Add the appropriate volume pre-diluted anti-PE 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 of pre-diluted Nanobeads for separating 1 x 108 cells in 1 ml of MojoSort™ Buffer. 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.
?. Add the 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 to the column through a 30 µm filter and collect the fraction containing the unlabeled cells.
?. [Example of magnetic separation with medium capacity columns:]
Wash the cells in the column 3 times with 3 mL of buffer and collect the fraction containing the unlabeled cells. Combine with the collected fraction from step 3. These cells may be useful as controls, to monitor purity/yield, or other purposes.
?. [Example of magnetic separation with medium capacity columns:]
Take 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. These are the positively isolated cells of interest; do not discard. To increase the purity of the magnetically labeled fraction repeat the isolation process with a new, freshly prepared column.