MojoSort™ Human anti-APC Nanobeads Column Protocol
- 1BioLegend
- BioLegendTech. support email: tech@biolegend.com
External link: https://www.biolegend.com/protocols/mojosort-human-anti-apc-nanobeads-column-protocol/4758/
Protocol Citation: Sam Li . MojoSort™ Human anti-APC Nanobeads Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7afhibn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: September 11, 2019
Last Modified: September 11, 2019
Protocol Integer ID: 27687
Keywords: MojoSort™, APC, nanobeads, column, cell separation
Abstract
BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.
Note: Due to the properties of our beads, it may be possible to use far fewer beads than with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service (tech@biolegend.com) if further assistance is needed.
Guidelines
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™.
Materials
MATERIALS
Human TruStain FcX™BioLegendCatalog #422301
MojoSort™ BufferBioLegendCatalog #480017
MojoSort™ Human anti-APC NanobeadsBioLegendCatalog #480089
APC-Conjugated Anti-Human AntibodyBioLegend
Additional reagents:
-commercially available cell separation columns
-5 mL polypropylene tubes
-70 µm cell strainer
Prepare cells from your tissue of interest or blood without lysing erythrocytes.
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.
Filter the cells with a 70 µm cell strainer, centrifuge at 300xg for 5 minutes, and resuspend in a small volume of MojoSort™ Buffer. Count and adjust the cell concentration to 1 x 108 cells/mL by adding MojoSort™ Buffer.
5m
Aliquot 100 µL of cell suspension (107 cells) into a new tube. Add 5 µL 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, add 50 µL of Human TruStain FcX™ 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.
1m
Check the recommended usage for flow cytometric staining of the APC-conjugated antibody indicated in the antibody technical datasheet. Calculate the volume to stain 107 cells (or desired amount of cells). Add the appropriate volume of pre-diluted APC-conjugated antibodyto the cell suspension, mix well and incubate on ice for 15 minutes.
Note: For the APC-conjugated antibodies, we recommend to do a titration to determine the optimal concentration
15m
Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
5m
Discard the supernatant and resuspend cells in 100 µL of MojoSort™ Buffer.
Vortex the anti-APC Nanobeads (to resuspend) at max speed, 5 touches, and prepare the dilutions to test. Add the appropriate volume of pre-diluted anti-APC Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, if the volume of pre-diluted Nanobeads for 1x107 cells is 10 µL, add 100 µL of pre-diluted 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.
Note: The amount of Nanobeads to use always depends on the frequency of the target, among a few other factors. We recommend to do a titration to determine the optimal concentration.
15m
Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
5m
Discard the supernatant.
Add the appropriate amount of MojoSort™ Buffer and proceed to separation. At least 500 µL is needed for column separation.
Note: There are several types of commercially available columns, depending on your application. Choose the one that fits best your experiment:
Example of magnetic separation with medium capacity columns:
Example of magnetic separation with medium capacity columns:
Place the column in a magnetic separator that fits the column.
Rinse the column with 3 mL of cell separation buffer.
Add the labeled cell suspension to the column through a 30 µm filter and collect the fraction containing the unlabeled cells.
Wash the cells in the column 3 times with 3 mL of buffer and collect the fraction containing the unlabeled cells. Combine with the collected fraction from step 3. These cells may be useful as controls, to monitor purity/yield, or other purposes.
Take away the column from the magnet and place it on a tube. Then add 5 mL of buffer and flush out the magnetically labeled fraction with a plunger or supplied device. These are the positively isolated cells of interest; do not discard. To increase the purity of the magnetically labeled fraction repeat the isolation process with a new, freshly prepared column.