Cell-Vive™ GMP MojoSort™ Human NK cell Isolation Kit

Kit Details

Kit Contents

For Cat# 480215:

• 200 μL Biotin-Antibody Cocktail
• 200 μL Streptavidin Nanobeads

 

For Cat# 480216:

• 2 vials of 1 mL Biotin-Antibody Cocktail
• 2 vials of 1 mL Streptavidin Nanobeads

 

 

Product Details

Reactivity

Human

Formulation

Cocktail: Phosphate-buffered solution containing 0.5% recombinant HSA and stabilizer

Particle: Aqueous solution containing 0.3% recombinant HSA

Endotoxin Level

< 0.1 EU/Test (individual components < 5 EU/mL)

Preparation

Serum-free antibodies were purified by affinity chromatography and conjugated with biotin under optimal conditions in a GMP facility. Magnetic nanobeads were conjugated to streptavidin under optimized conditions in a GMP facility. Contains no preservatives.

Storage & Handling

Undiluted at 4°C (2°C-8°C)

Application

Cell Separation - Quality tested

Recommended Usage

10 μl of Biotin-Antibody Cocktail for 1 x 10⁷ PBMCs in 100 μl separation buffer.

10 μl of Streptavidin Nanobeads for 1 x 10⁷ PBMCs in 100 μl separation buffer.

 

Antibody or cocktail dilution to use in column: 4x

Nanobead dilution to use in column: 6x

Application Notes

This kit is optimized for the negative isolation of NK cells from human peripheral blood mononuclear cells (PBMCs). Alternate sources of cell suspensions may require protocol optimization.

 

Recommended protocol for negative isolation of NK cells from human PBMCs

 

Isolate PBMCs using density gradient centrifugation. If working with another tissue, prepare a single cell suspension according to standard protocols. Excess dead or red blood cells may increase non-specific binding and removal prior to separation may increase performance. Filter the cell suspension with a sterile 40 µm nylon cell strainer for optimal performance.

 

Use sterile consumables and work in a biosafety cabinet. To maintain cell viability and reduce non-specific binding, work quickly, keep cells cold, and use pre-chilled separation buffer kept on ice throughout the procedure.

 

Separator Protocol

 

This procedure is optimized at 1 x 10⁷ cells/test. When working with more cells, optimize the conditions to your specific cell number and tissue for best results.

1. Resuspend cells at 1 x 10⁸ cells/mL with Cell-Vive™ CD Cell Separation Buffer, GMP (Cat# 420512) or other commercially available separation buffer.

2. Aliquot 100 μL (10⁷ cells) into appropriate tube.

   Optional: Reserve an aliquot of unseparated cells as a control.

   1. Scale up to 2 mL (2 x 10⁸ cells) in 12 x 75 mm (5 mL) polypropylene tube for 5 mL magnet (Cat# 480019).
   2. Scale up to 10 mL (1 x 10⁹ cells) in 17 x 100 mm (14 mL) polypropylene tube for 14 mL magnet (Cat# 480020).
3. Add 10 μL of the biotin-antibody cocktail per 10⁷ cells. Immediately, mix well to fully incorporate, and incubate on ice for 15 minutes.
4. Wash the sample with separation buffer and gently mixing. Centrifuge the cell suspension at 300xg for 5 minutes and discard the supernatant. Resuspend well in 100 μL per 10⁷ cells.
5. Resuspend the streptavidin nanobeads well by vortexing at maximum speed, at least 5 touches. Add 10 μL of streptavidin nanobeads per 10⁷ cells. Immediately, mix well to fully incorporate, and incubate on ice for 15 minutes.
6. Add separation buffer up to indicated volume. Mix well by pipetting to fully incorporate.
   1. 3.5 mL for separating ≤ 2 x 10⁸ cells using the 5 mL magnet.
   2. 5 mL for separating ≤ 5 x 10⁸ cells, or 10 mL for separating 5 x 10⁸ - 1 x 10⁹ cells, using the 14 mL magnet.
7. Place the tube in the MojoSort™ magnet to perform separation.
   1. 5 minutes separation time for 5 mL magnet.
   2. 10 minutes separation time for 14 mL magnet.
8. Carefully, pour out the unlabeled fraction into a collection tube. If these are your cells of interest, do not discard, place tube on ice. Remove tube containing magnetically labeled fraction from the magnet, do not discard.
9. Repeat steps 6-8 on the magnetically labeled fraction for a total of 2 separations for optimal purity. Pool the unlabeled negative fractions and keep the labeled cells for downstream applications. The fraction that is not of interest may be useful as staining controls, to monitor purity/yield, or other purposes.

 

Column Protocol

 

Prepare biotin-antibody cocktail and streptavidin nanobeads at dilutions recommended for column use found on the TDS. Vortex streptavidin nanobeads well at maximum speed to resuspend before diluting with Cell-Vive™ CD Cell Separation Buffer, GMP (Cat# 420512) or other commercially available separation buffer.

 

Cat# 480215/480216 recommended antibody cocktail dilution to use on columns: 4x. Recommended streptavidin nanobead dilution to use on columns: 6x

 

There are several types of commercially available columns, depending on your application. Choose the one that best fits your experiment. Column cell capacity is dependent on column volume, target cell abundance, type, and size. NK cell abundance within PBMCs can vary significantly between donors. To avoid overloading columns, we do not recommend loading the maximum labeled cell capacity. For samples with high cell numbers, split the sample across multiple columns and pool the fractions after separation. Protocols should be optimized empirically for use on different column systems.

 

1. Resuspend cells at 1 x 10⁸ cells/mL with separation buffer.

   Optional: Filter cell suspension through a 40 μm filter.

2. Aliquot 100 μL (10⁷ cells) into appropriate tube.

   Optional: Reserve an aliquot of unseparated cells as a control.

   Note: Scale up volumes for larger cell numbers dependent on column capacity. For example, add 100 μL antibody cocktail and nanobeads for 1 x 10⁸ cells (equivalent to 10 tests at 1 x 10⁷ cells/test) in 1 mL separation buffer.

3. Add 10 μL of the diluted biotin-antibody cocktail per 10⁷ cells. Immediately, mix well to fully incorporate, and incubate on ice for 15 minutes.
4. Wash the sample with separation buffer and gently mixing. Centrifuge the cell suspension at 300xg for 5 minutes and discard the supernatant. Resuspend well in 100 μL per 10⁷ cells.
5. Resuspend the diluted streptavidin nanobeads well by vortexing at maximum speed, at least 5 touches. Add 10 μL of streptavidin nanobeads per 10⁷ cells. Immediately, mix well to fully incorporate, and incubate on ice for 15 minutes.
6. Wash the sample with separation buffer and gently mixing. Centrifuge the cell suspension at 300xg for 5 minutes and discard the supernatant.

7. Resuspend cells well in 1 mL separation buffer by pipetting and place on ice.

   Note: We recommend a maximum cell density of 1 x 10⁸ million cells/mL of buffer (equivalent to 10 tests/mL of buffer) for column loading. A lower cell density may improve recovery. If working with samples ≥ 5x10⁷ cells, verify the maximum labeled cell capacity of the column. To avoid overloading, the sample may need to be split over multiple columns and pooled after separation.

8. Prepare the column by placing in the appropriate corresponding magnetic separator and rinsing with 3 mL separation buffer. Discard the flow through.

9. Place an appropriate tube below the column to collect the negative fraction. Add the cell suspension to the column and collect the fraction containing the unlabeled cells.

   Recommended: Filter cell suspension through a sterile 40 μm filter to remove aggregates before adding to the column.

10. Wash the column with 3 mL separation buffer. Collect the flow-through containing the unlabeled cells and pool with unlabeled fraction collected at step 9. If these are the cells of interest, do not discard. Place tube containing unlabeled cells on ice.

    Optional: Performing additional washes on column may increase target cell recovery.

11. Remove the column from the magnet and place it in a collection tube. Allow the column to demagnetize for 1-2 minutes. Add 5 mL separation buffer to the column reservoir and flush the magnetically labeled fraction into the collection tube with a plunger or supplied device. This contains the magnetically labeled cells, this fraction may be useful as controls, to monitor purity/yield, or other purposes.

Disclaimer

BioLegend Cell-Vive™ GMP MojoSort™ cell separation tools are for research use only. Suitable for ex vivo cell processing use. Not for use in diagnostic or therapeutic use. Not for resale. BioLegend will not be held responsible for patent infringement or other violations that may occur with the use of our products.

Antigen Details

Gene ID

NA

UniProt

View information about NK on UniProt.org

Documentation

• Technical data sheet
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Certificate of Analysis