Isolate Virtually Any Cell Type Using EasySep™ or EasySep™ Release Indirect Selection Kits
- 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/FITC/PE/APC-conjugated antibody. The desired final concentration will be the lowest concentration of your primary antibody that gives the greatest fluorescence intensity shift by flow cytometry (for best results, aim to achieve 2 log staining intensity above background). We suggest using a final concentration of primary antibody between 0.3 - 3.0 µg/mL of cells, the optimal concentration will vary depending on the starting frequency of your cells of interest.
- <10%, use a final concentration of antibody between 0.3 - 0.5 µg/mL
- 10-40%, use a final concentration of antibody between 0.5 - 1.0 µg/mL
- 40-60%, use a final concentration of antibody between 1.0 - 2.0 µg/mL
- >60% use a final concentration of antibody between 2.0 - 3.0 µg/mL *
*NOTE: For samples where the start percentage of desired cells is greater than 60%, it may be advantageous to reduce the number of magnetic separations in order to improve recovery.
- 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).