Optimization of ·¡²¹²õ²â³§±ð±èâ„¢ Releasable RapidSpheresâ„¢ Indirect Positive Selection Kits
Technical tip from our dedicated team of Product and Scientific Support specialists
ºÚÁÏ³Ô¹Ï offers ·¡²¹²õ²â³§±ð±èâ„¢ Release indirect selection kits to isolate virtually any cell type. The kits are designed to isolate cells that are labeled with biotinylated, APC- or PE-conjugated antibodies (not provided). The labeled cells are then targeted with antibody complexes recognizing biotin, APC or PE and Releasable RapidSpheresâ„¢ and are separated using an ·¡²¹²õ²â³§±ð±èâ„¢ 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 ·¡²¹²õ²â³§±ð±èâ„¢-isolated, biotin-, APC- or 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-, APC- or PE-conjugated antibody and associated ·¡²¹²õ²â³§±ð±èâ„¢ Release Biotin, APC or PE Positive Selection Cocktail according to the following reagent matrix:
- Recovery of positively selected cells is dependent on the quality of biotinylated, APC 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 ·¡²¹²õ²â³§±ð±èâ„¢ Release kit, antibody complexes remain bound to the cell surface and may interact with Brilliant Violetâ„¢ antibody conjugates, polyethylene glycol-modified proteins, or other chemically related ligands. If Brilliant Violetâ„¢ antibody conjugates are used, these should be carefully titrated on ·¡²¹²õ²â³§±ð±èâ„¢ Release-isolated cells prior to analysis by flow cytometry or fluorescence microscopy. For purity assessment with Brilliant Violetâ„¢ antibody conjugates, use of BD Horizon Brilliantâ„¢ Stain Buffer is recommended to reduce non-specific interactions.
- 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).
·¡²¹²õ²â³§±ð±èâ„¢ Release Biotin
·¡²¹²õ²â³§±ð±èâ„¢ Release PE and APC
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. Concentration of ·¡²¹²õ²â³§±ð±èâ„¢ Releasable RapidSpheresâ„¢ 50201 is kept at 100uL/mL in all conditions.
For further assistance and information, please contact techsupport@stemcell.com.
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