MagniSort™ Frequently Asked Questions
Q: Mouse secondary organs and human peripheral blood mononuclear cells are listed in the manual under cell preparation. Is this kit also compatible with whole blood?
No, the MagniSort™ kits are not compatible with whole blood samples.
Q: Are these kits compatible with non-lymphoid tissues?
These kits have not been validated in non-lymphoid tissues. However, for positive selection kits, if the non-lymphoid tissues have been processed and prepared to enrich for lymphocytes (with Ficoll®, for example), the kits can be used. Depending on the frequency of the target cell in this enriched population, the amount of the magnetic beads can be titrated to optimize purity. Negative selection kits are not recommended for non-lymphoid tissues.
Q: I currently use flow cytometry (FACS) for sorting cells. What is the advantage of magnetic cell separation compared to FACS sorting?
Magnetic cell separation kits are easy to use, and the time to result is much faster than FACS sorting, especially when a large number of cells needs to be sorted or when multiple samples need to be sorted individually. In addition, cells are maintained under normal physiological conditions during the magnetic separation process without being pressurized or being charged. Magnetic cell separation kits are also an excellent choice for enriching for very rare cells before FACS sorting.
Q: There are two types of kits – positive and negative selection. When would you choose one over the other?
The choice between positive and negative selection is best determined by the end users as it is dependent on their specific needs. Cells from both positive and negative selection are viable and can respond to further stimulation.
Q: Does magnetic sorting have an effect on cell viability?
Cells immediately following magnetic sorting have the same viability compared to unsorted cells.
Q: What does yield and purity mean? How are each of these parameters calculated?
Yield is the percentage of target cells harvested based on the number of cells theoretically available in the starting population. To calculate yield, stain a sample of unsorted and sorted cells with antibodies to identify the target cells, and count the total number of target cells using 123count eBeads™ (cat. 01-1234) on the cytometer. Yield = # of sorted target cells / # of target cells unsorted.
Purity is the percentage of cells harvested that are target cells. Purity can be directly generated by flow cytometric analysis of sorted samples. To obtain purity, stain the sorted cells with antibodies to identify the target cells and analyze by flow cytometry. Gate on live, single cells, and subsequently gate on the target cell population. Purity is the percentage of target cells in the live, single-cell gate.
Q: How can I analyze the purity of a sample?
To analyze the purity of a sample, stain a sample of sorted cells with antibodies to identify the target cells (see Technical Data Sheet for each kit for recommendation of clones) and analyze by flow cytometry.
Q: I have sticky cells. What buffer do you recommend to prevent the cells from clumping?
For magnetic cell separation, we recommended PBS or HBSS buffers supplemented with 3%-10% fetal bovine serum and 10 mM EDTA. EDTA will help prevent the cells from clumping. Media such as RPMI and DMEM are not recommended because they interfere with the performance of the kits. Thorough mixing of the cells and filtration through nylon filters are also recommended to ensure single-cell suspension and removal of debris.
Q: I have cells in culture. Do I need to wash the cells and remove the culture media prior to starting the MagniSort protocol?
Cells can be stained with the antibody or antibody cocktails in the culture media; however, subsequent washes should be performed with the recommended cell separation buffers before the addition of magnetic beads. Culture media such as RPMI and DMEM interfere with the binding of magnetic beads to target cells.
Q: How does the binding of the beads affect the cells? Is the function of positively selected cells altered by the bound particles/antibodies?
The binding of the positive selection beads does not affect the viability or function of bound cells. Some minor changes in light scatter may be observed during flow cytometric analysis, depending on the cell type.
Q: Can the magnetic beads be removed from the cells after enrichment?
For positive selection, the magnetic beads cannot be removed; however, studies have shown that removal of the beads is not necessary for subsequent applications. For negative selection, the target cells are untouched and not bound to magnetic beads.
Q: Can the cells be stained and/or sorted after enrichment?
Q: Can the cells incubate longer with the antibody/cocktail than the recommended 10 minutes?
Incubation of cells with antibody for longer than 10 minutes at room temperature will not reduce the performance of the kit as long as the viability of the cells is not affected. If users intend to incubate for longer than 30 minutes, we recommend incubating at 2-8°C. We do not recommend incubating with magnetic beads for longer than the recommended time.
Q: Are the beads in the MagniSort kit compatible with other commercially available magnets?
MagniSort beads are compatible with STEMCELL Technologies’ EasySep magnet. We do not recommend use with other column-based or column-free magnets.
Q: In the protocol, it states that the maximum cell number is 2x108 cells. Is there a minimum cell number?
We do not recommend starting with fewer than 5x106 cells as this may result in poor purity or yield. For 5-10x106 cells, resuspend in 50 µL and adjust volumes of antibody and beads accordingly; for remaining steps, follow the protocol as written.
Q: If I have fewer cells than the minimum requirement, will this affect purity and yield?
We do not recommend starting with fewer than 5x106 cells as this may result in poor purity and/or yield. If starting with fewer than 5x106 cells, it may be possible to improve purity or yield by modifying the amount of magnetic beads added and should be determined empirically.
Q: Are the magnetic beads large, should I cut the tips of pipets to pipet the beads?
The magnetic beads are much smaller than eukaryotic cells, therefore, any pipet that can be used to pipet eukaryotic cells can be used to pipet the beads.
Q: I accidentally vortexed the vial of magnetic beads, will this affect purity and yield?
An isolated and accidental incident of vortexing should not affect performance. However, vortexing beads may lead to significant bubble formation and oxidation. We recommend avoiding extensive vortexing to ensure product stability and performance.
Q: What is the size of the magnetic beads? Will the presence of the beads affect the function of positively selected cells?
The magnetic beads are much smaller than eukaryotic cells; the exact size is proprietary. However, studies have shown that removal of the beads is not necessary for subsequent applications.
We’ve extensively investigated the effects of the beads on cell activation and proliferation. The bound beads do not activate cells in the absence of any additional stimulation. When stimulated, our positively sorted cells function the same as negatively selected cells. Additionally, there is minimal effect on FSC and SSC, and the beads cannot be seen on positively sorted cells by light microscopy.
Q: Why is the pellet of desired cells slightly brown after positive and/or negative selection?
After positive selection, the magnetic beads that are attached to the desired cells post-sort can cause the cell pellet to appear brown. However, extensive functional studies have shown that these attached beads do not activate cells without further stimulation, and do not inhibit further activation of the cells.
After negative selection, some residual free particles may not be completely removed by the magnet, leading to a slight brown tint of the cell pellet. This will not affect cell viability or function. However it is possible to remove these residual magnetic beads as follows: resuspend the cells in 2.5 mL of the recommended cell separation buffer in a 12x75 mm tube and place into the magnet for 5 minutes, then pour the desired cells into a new tube; the brown tint should be removed. This process may slightly reduce the recovery of cells of interest.