Optimization of EasySep™ Releasable RapidSpheres™ Indirect Positive Selection Kits
STEMCELL Technologies offers EasySep™ Release indirect selection kits to isolate virtually any cell type. The kits are designed to isolate cells that are labeled with biotinylated or PE-conjugated antibodies (not provided). The labeled cells are then targeted with antibody complexes recognizing biotin or PE and Releasable RapidSpheres™ and are separated using an EasySep™ magnet without the use of columns. Unwanted cells are simply poured off, while desired cells remain in the tube. In a final step, bound magnetic particles are removed from the EasySep™-isolated, biotin or PE-antibody labeled cells, which are immediately available for downstream applications such as flow cytometry, culture, or DNA/RNA extraction.
*For use with other species, we recommend adding a species-specific blocker to cell the suspension to minimize non-specific binding. For more information on using Fc receptor (FcR) blockers, please refer to our Technical Tip: Importance of Using an FcR Blocker During Cell Separation Procedures.
The recommended protocols outlined on the Product Information Sheet (PIS) for each kit have been optimized for use with fresh or previously frozen human peripheral blood mononuclear cells (PBMCs), washed leukapheresis samples, or mouse splenocytes, across a variety of cell surface markers. Further protocol optimization may be required depending on your specific application or starting sample. Here are some protocol optimization tips:
- Ensure cells are in single-cell suspension.
- Add species-specific blocker to cell suspension to minimize non-specific binding. For more information on using Fc receptor (FcR) blockers, please refer to our Technical Tip: Importance of Using an FcR Blocker During Cell Separation Procedures.
- We recommend titrating your primary biotin- or PE-conjugated antibody and associated EasySep™ Release Biotin or PE Positive Selection Cocktail according to the following reagent matrix:
Primary AntibodyPositive Selection Cocktail2ug/mL (PE) or 3ug/mL (Biotin)100 µl/mL1ug/mL (PE or Biotin)50 µl/mL0.5ug/mL - 0.3 ug/ml (PE or Biotin)25 µl/mL
Each reagent test condition outlined in the reagent matrix should be performed with and without a wash step following the primary antibody labeling step, for a total of 6 test conditions.
- Recovery of positively selected cells is dependent on the quality of biotinylated or PE-conjugated antibody (not provided) used for positive selection. Antibodies that have expired or that have been stored improperly may show lower affinity for the surface marker on the target cell, resulting in lower recovery.
- Following cell isolation with this EasySep™ Release kit, antibody complexes remain bound to the cell surface and may interact with Brilliant Violet™ antibody conjugates, polyethylene glycol-modified proteins, or other chemically related ligands.
- Some antibodies used to assess purity of the isolated cells will be blocked by the initial selection antibody, in which case you should stain with an alternate antibody. You could use an antibody for a different cell surface antigen (i.e. assess purity using alternate marker expressed on your target cells), or use a fluorochrome-conjugated antibody that will recognize the primary antibody (i.e. a goat anti-mouse antibody, such as GAM-FITC).
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