Identification of human myeloid-derived suppressor cell (MDSC) subpopulations

This application protocol describes an exemplary procedure, which can be used to identify human MDSC subpopulations. In this protocol, MDSCs were detected in PBMCs prepared from fresh blood samples of healthy donors.

Protocol

  • Volumes given below are optimized for staining of up to 107 nucleated cells. When working with fewer than 107 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×107 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes). 
  1. Determine cell number. For each biological sample, prepare two tubes containing up to 107 nucleated cells each. 
  2. Centrifuge tubes containing cell suspension at 300×g for 10 minutes. Aspirate supernatants completely. 
  3. Resuspend up to 107 nucleated cells per 200 μL of buffer. 
  4. Add 20 μL of MDSC Staining Cocktail to the first tube. 
  5. Add 20 μL of MDSC Control Cocktail to the second tube. 
  6. (Optional) In case of analysis based on extra markers for pre-gating (e.g. CD66b or CD15): Add 4 μL of the optional APC-conjugated antibody to each of both tubes. For a more detailed phenotypical analysis of PMN-MDSC subsets (e.g. LOX-1, CD10): Add 4 μL of APC-conjugated antibody to the tube containing the MDSC Staining Cocktail and 4 μL of REA Control (S)-APC to the tube containing the MDSC Control Cocktail. 
  7. Mix well and incubate for 10 minutes in the dark in the refrigerator (2–8 °C). 
    Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times. 
  8. Wash cells by adding 1–2 mL of buffer per 107 cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely. 
  9. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry. 
    Note: Store samples at 2–8 °C protected from light until analysis. 
  10. Proceed to flow cytometric analysis.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.
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