MojoSort™ Whole Blood Human NK Cell Isolation Kit Protocol

Reagent List

MojoSort™ Buffer (5X) (Cat. No. 480017) MojoSort™ Magnet (Cat. No. 480019/480020) or compatible magnetic separation system separation system Adjustable pipettes Serological pipet

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™.

 

Protocol
 

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Product description and procedure summary:
MojoSort™ Whole Blood isolation kits allow for enrichment of untouched cells of interest directly from whole blood, while magnetically depleting out other cell types typically present in human whole blood. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.

Note: This procedure is optimized using 1mL whole blood per tube. Scale volumes in this procedure accordingly, e.g. using 2 mL whole blood, use twice the amount of antibody cocktail and nanobeads. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. Scale up volumes if using 14 mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

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1. Collect whole blood in collection tube that has anticoagulant, preferably EDTA.
   Note: Keep MojoSort™ Buffer on ice throughout the procedure.
    
2. Aliquot 1mL of human whole blood into a 5mL (12 x 75 mm) polypropylene tube. Add 20µL of the Biotin-Antibody Cocktail. Mix well and incubate at room temperature for 5 minutes. Scale up or down the volume accordingly if separating more blood. For example, add 40µL of biotinylated antibody cocktail for separating 2mL blood. If using the 14mL magnet system, it is recommended to do the incubation on a rocker or to invert the tube every minute during incubation.
    
3. Resuspend the beads by vortexing, maximum speed, 5 touches. Add 20 µL of Streptavidin Nanobeads. Mix well and incubate at room temperature for 5 minutes. Scale up the volume accordingly if separating more volume of whole blood. For example, add 40 µL of Nanobeads for separating 2 mL of whole blood. If using the 14mL magnet system, it is recommended to do the incubation on a rocker or to gently invert the tube every minute during incubation.
    
4. 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 NK Cell Isolation Protocol: Separation Volumes and Magnet Times
   Sample's Volume:	MojoSort™ Buffer Volume	Magnet	Magnet Incubation Time
   ≤ 1 mL	Top volume up to 3.5mL for separation (e.g. for 1 mL sample add 2.5mL buffer for the first separation, then add 3.5mL buffer for subsequent separations)	MojoSort™ magnet (cat: 480019)	5 minutes
   1 – 5 mL	Top volume up to 10mL for each separation (e.g. for 5mL sample add 5mL buffer for the first separation, then add 10mL buffer for subsequent separations)	MojoSort™ magnet 14mL (480020)	10 minutes

    
5. 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.
    
6. Pour out the unlabeled fraction. If these are your cells of interest, DO NOT DISCARD. Resuspend the labeled cells in MojoSort™ Buffer.
    
7. Repeat steps 4-6 on the labeled fraction twice more for a total of 2 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.
    
8. Resuspend the magnetically labeled cells in desired volume, making sure to rinse the sides of the tube for better recovery.
    
9. (Optional) Lyse remaining erythrocytes using 20 mL room temperature 1 X Red Blood Cell Lysis Buffer (10X Cat. No. 420301 / 420302). Incubate 15min, in dark at room temperature followed by filtering through a cell strainer (40-70m). Wash twice with MojoSort™ Buffer

 

Chart

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.