TITLE: MojoSort™ Human CD14+ Monocytes Isolation Kit Protocol
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Product description and procedure summary:</span><span> This kit is designed for the isolation of untouched CD14</span><span style = "vertical-align:super;">+</span><span>CD16- monocytes from peripheral blood mononuclear cells (PBMCs). Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads. 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.</span></div><div class = "text-block"><span style = "font-weight:bold;">
</span><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 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.</span><span></span></div></div>

[STEPS]
?. [Platelet Removal Protocol]
Dilute blood with 2-4 times (volume/volume) 1X PBS.
?. [Platelet Removal Protocol]
Carefully layer diluted blood over 12.5mL of isolation medium in a 50mL tube.
?. [Platelet Removal Protocol]
Centrifuge at 400xg for 25 minutes at room temperature in a swinging-bucket rotor without the brake.
?. [Platelet Removal Protocol]
Aspirate the upper layer of the gradient (serum), leaving the interphase containing the mononuclear cells undisturbed.
?. [Platelet Removal Protocol]
Carefully transfer the mononuclear cells to a new 50mL tube.
?. [Platelet Removal Protocol]
Fill the tube with 1X PBS, mix, and centrifuge at 200xg for 8 minutes at room temperature. Carefully remove supernatant as much as possible.
?. [Platelet Removal Protocol]
Repeat step 6.
?. [Platelet Removal Protocol]
Proceed to separation protocol.
?. [Separation Protocol]
In the final wash of your sample preparation, resuspend the cells in MojoSort™ Buffer by adding up to 4mL in a 5 mL (12 x 75 mm) polypropylene tube.Note: Keep MojoSort™ Buffer on ice throughout the procedure.
?. [Separation Protocol]
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 108 cells/mL.
?. [Separation Protocol]
Aliquot 100 µL of cell suspension (107 cells) into a new tube. Add 5µL 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, if the volume of Human TruStain FcX™ for 1x107 cells is 5µL, add 50µL for 1 x 108 cells. When working with less than 107 cells, use indicated volumes for 107 cells.
?. [Separation Protocol]
Add10µL of the Biotin-Antibody Cocktail.Mix well andincubate 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 108 cells in 1 ml of MojoSort™ Buffer. When working with less than 107 cells, use indicated volumes for 107 cells.Optional: Take an aliquot before adding the cocktail to monitor purity and yield.
?. [Separation Protocol]
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 108 cells in 1 ml of MojoSort™ Buffer. When working with less than 107 cells, use indicated volumes for 107 cells.
?. [Separation Protocol]
Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes.
?. [Separation Protocol]
Discard supernatant.
?. [Separation Protocol]
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.
?. [Separation Protocol]
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.
?. [Separation Protocol]
Pour out and collect the liquid. These are your cells of interest; DO NOT DISCARD. Resuspend the labeled cells in 2.5mL MojoSort™ Buffer.
?. [Separation Protocol]
Repeat steps 8-10 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.