MojoSort™ Mouse CD90.2 Selection Kit Protocol

Reagent List

MojoSort™ Human CD45 Selection Kit MojoSort™ Buffer (5X) (Cat. No. 480017) MojoSort™ Magnet (Cat. No. 480019/480020) or compatible magnetic separation system Adjustable pipettes 70µm filters (one per sample)

5mL (12 x 75mm) or 14mL (17 x 100mm) polypropylene tubes Reagents for sample preparation Reagents and instruments (Flow cytometer) to determine yield and purity

 

Important Note

MojoSort™ magnetic particles can be used with other commercially available magnetic separators, both free standing magnets and column-based systems. Because MojoSort™ protocols are optimized for the MojoSort™ separator, the protocols may need to be adjusted for other systems. Please contact BioLegend Technical Service (tech@biolegend.com) for more information and guidance. We do not recommend using MojoSort™ particles for BD’s IMag™ or Life Technologies’ DynaMag™. A separate protocol for the use of this kit on column-based magnetic separators is available (list of protocols adapted for column use available here: biolegend.com/mojosort/columnIndex)

 

Protocol

Product description and procedure summary: 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.

 

Note:  This procedure is optimized for the isolation of 10⁷ to 2 x 10⁸ cells per tube. If working with fewer than 10⁷ cells, keep volumes as indicated for 10⁷ 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. Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

 

1. Prepare cells from your tissue of interest or blood without lysing erythrocytes.
    
2. 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.

 

3. 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.

 

4. Aliquot 100µL of cell suspension (10⁷ cells) into a new tube. Add 10µL of TruStain FcX™ PLUS (anti-mouse CD16/32 antibody), 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 Mouse TruStain FcX™ PLUS for 1x10⁷cells is 10µL, add 100µL for 1 x 10⁸ cells. When working with less than 10⁷ cells, use indicated volumes for 10⁷cells.
   
   Note: older lots of MojoSort™ Mouse CD90.2 Selection Kits may not contain TruStain FcX™ PLUS as a part of the kit. If using an older lot that does not contain this reagent, skip this step

 

5. 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 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.

 

6. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes.

 

7. Discard supernatant and resuspend in 100µL of MojoSort™ Buffer.

 

8. 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.

 

9. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes.

 

10. Discard supernatant.

 

11. 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.

 

12. 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.

 

13. Pour out the unlabeled fraction. If these are your cells of interest, DO NOT DISCARD. Resuspend the labeled cells in 2.5mL MojoSort™ Buffer. 

 

14. Repeat steps 11-13 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.

 

 

Schematic Protocol:

Application notes: To use this product in magnetic separation columns, a titration of the Nanobeads should be performed. Optimal concentration for magnetic separation columns is lot-specific. Please contact BioLegend Technical Service (tech@biolegend.com) for further assistance on how to use MojoSort™ Nanobeads in magnetic separation columns.