MojoSort on Columns™ Isolation Kits Column Protocol – A

 

Introduction

 

BioLegend’s MojoSort on Columns™ Cell Isolation kits are compatible with our Large Columns (Cat. No. 480178) as well as columns from other suppliers. Please contact BioLegend Technical Service for more information and guidance on compatibility with 3rd party column systems.

 

To ensure you are referencing the correct protocol for your cell separation product, please see the Related Protocols section of your kit-specific Technical Datasheet.

 

Procedure Summary:

 

Following this protocol, your target cells remain unlabeled and flow through the column while the undesired labeled cells are magnetically retained in the column matrix. First, your starting sample, such as whole blood or Leukopak-derived PBMCs, are incubated with the biotin-labeled antibody cocktail followed by incubation with magnetic Streptavidin Nanobead particles. Then, your sample is transferred to a column placed in a magnetic column separator. The separator establishes a strong magnetic field within the column matrix, allowing the bead-labeled cells to be retained while the untouched cells flow through for collection. These untouched cells are your population of interest. The magnetically labeled cells, which are retained in the column, may also be eluted as a control sample and collected. Downstream applications include functional assays, gene expression analysis, phenotypic characterization, and more.

 

Important Notes:

• One test is defined as cell separation from 1 x 10⁷ cells.
• This procedure is optimized for the isolation of 1 x 10⁷ cells/test. If working with fewer than 1 x 10⁷ cells, keep volumes as indicated for 1 x 10⁷ cells. When working with more cells per sample, optimize the conditions to your specific cell number and tissue by scaling appropriately for best results.
• Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.
• Our kits designed for human samples are optimized for PBMCs isolated from fresh whole blood and/or frozen PBMCS derived from Leukopaks. Please see product-specific technical datasheet to determine optimized sample types for the kit of interest. Ensure cells are prepared using an appropriate method suited to your sample type.
• Keep MojoSort™ Buffer on ice throughout the procedure.

 

Reagent list

• MojoSort™ Buffer (5X) (Cat. No. 480017)
• PBS without Ca²⁺/Mg²⁺
• MojoSort on Columns™ - Multistand (Cat. No. 480177) or other equivalent
• MojoSort on Columns™ - Large Columns (Cat. No. 480178) or other equivalent
• MojoSort on Columns™ - Separator for Large Columns (Cat. No. 480179) or other equivalent
• Tube for cell samples such as 5 mL (12 x 75 mm) or 14 mL (17 x 100 mm) polypropylene tubes
• Adjustable pipettes
• 30 µm or 40 µm cell strainer
• Reagents for sample preparation and/or cell culture
• Reagents and instruments to determine yield and purity (e.g. flow cytometer)

 

Protocol

 

Sample Preparation:

 

1. Resuspend your cells in 1X MojoSort™ Buffer to an appropriate volume for cell filtration.
2. Filter the cell suspension through a 30 µm or 40 µm cell strainer into a new tube. Centrifuge at 300 x g for 5 minutes and resuspend the pellet in an appropriate volume of MojoSort™ Buffer and count the cells.
3. Adjust the cell concentration to 1 x 10⁸ cells/mL.

Antibody Labeling:

 

4. Aliquot 100 µL of the cell suspension (equivalent to 1 test at 1 x 10⁷ cells/test) into a new tube. Add 10 µL of the Biotin-Antibody Cocktail, mix well by gently tapping the tube, and incubate on ice for 15 minutes.
   Note: Scale up the volumes as needed for larger cell numbers. For example, add 100 µL of the Biotin-Antibody Cocktail for 1 x 10⁸ cells (equivalent to 10 tests at 1 x 10⁷ cells/test) in 1 mL of MojoSort™ Buffer. When processing fewer than 1 x 10⁷ cells, use the indicated volumes for 1 test (1 x 10⁷ cells).

Nanobeads Labeling:

 

5. Resuspend the Streptavidin Nanobeads by vortexing at maximum speed with five quick touches. Add 10 µL of the Nanobeads to the cell suspension, mix well by gently tapping the tube and incubate on ice for 15 minutes. During the incubation, you may set up the column and system (step #7).
   Note: Scale up the Nanobead volumes as needed for larger cell numbers. For example, add 100 µL of the Nanobeads for 1 x 10⁸ cells (equivalent to 10 tests at 1 x 10⁷ cells/test) in 1 mL of MojoSort™ Buffer. When processing fewer than 10⁷ cells, use the indicated volumes for 10⁷ cells.
6. After incubation, add enough 1X MojoSort™ Buffer to your cell sample to bring up to 1 mL total volume. Leave on ice until you are ready to load cells into the column in step #9.
   Note: We recommend a max cell density of 200 million cells/mL of buffer (equivalent to 20 tests/mL of buffer) for column loading. A lower cell density may result in improved recovering/yield. Use may need to optimize density to desired cell type.

Fig. 1. MojoSort on Columns™ System Setup

 

 

System Setup:

 

7. During the incubation in step #5, you may set up your column system. Gently place the magnetic column separator onto the multistand. Then place a new sterile column into the designated slot in the separator.
   Note: Ensure the multistand is securely placed on a balanced and stable surface and the magnetic separator is level. Insert the column into the magnetic separator's designated slot, ensuring it is fully seated and secure.

Column Wash:

 

8. Only proceed to this column wash step once your cells from step #6 are ready in order to prevent the column from running dry. Gently place a collection tube under the column and rinse the column with 3 mL of 1X MojoSort™ Buffer. Once the buffer has drained into the tube, discard the flowthrough. Proceed immediately to the next step to avoid column dry out.
   Note: Ensure the buffer has completed flowing through the column reservoir before proceeding to next step. Please do not allow the column to run dry to avoid formation of air bubbles.

Unlabeled Cell Collection:

 

9. Place a new collection tube under the column. Gently transfer the labeled cell samples from step #6 into the column chamber. Collect the flow through fraction containing the unlabeled cells. These are the cells of interest; do not discard this negative fraction.
   Optional: If concerned about clumping in your sample, you may directly apply a cell strainer on top of the column chamber when loading the cells for an additional filter step.
   Note: We recommend a max cell density of 200 million cells/mL of buffer (equivalent to 20 tests/mL of buffer) for column loading. A lower cell density may result in improved recovering/yield. Use may need to optimize density to desired cell type.
   Note: If working with larger cell sample volumes, you may need to load cell sample in multiple and smaller volumes to avoid overloading the column. If so, do not let column run dry between loadings.
10. To further collect unlabeled cells, load and wash the column with an additional 3 mL of 1X MojoSort™ Buffer while collecting the flowthrough. These are the cells of interest; do not discard this negative fraction. Pool the flowthrough with your collection from step #9. Repeat two more times for a total of 3 wash steps.
11. You may spin down your pooled cells for resuspension and use in your downstream analysis or cell culture.

Optional - Labeled Cell Collection:

 

12. If desired, you may collect your labeled cells once the column has stopped dripping from step #10. Immediately recover the labeled cells by removing the column from the magnetic separator and place over a new collection tube. Allow the column to demagnetize for 1–2 minutes.
13. Add 5 mL of MojoSort™ Buffer and flush out the magnetically labeled cells using the plunger (plunger included with our columns).
14. You may spin down your labeled cells for resuspension and use as staining controls, for monitoring purity/yield, or other applications.