Flow cytometry staining protocol for CD321 antibodies (1:11)

Protocol for immunostaining using CD321 antibodies with recommended working dilution of 1:11

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Protocol

  • The recommended antibody dilution for labeling of cells and subsequent analysis by flow cytometry is 1:11 for up to 107 cells in final volume of 110 µL.
  • Volumes given below are for up to 106 nucleated cells. When working with fewer than 106 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 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes). 
  1. Determine cell number.
  2. Centrifuge cell suspension at 600×g for 10 minutes. Aspirate supernatant completely.
  3. Resuspend up to 107 nucleated cells per 100 µL of PBS.
  4. Add 10 µL of the antibody.
  5. Mix well and incubate for 10 minutes in the dark at 2-8 °C. 
  6. Wash cells by adding 1 mL PBS and centrifuge at 600×g for 10 minutes. Aspirate supernatant, leave 100 µL of supernatant to avoid unintended aspiration of cell pellet. If biotinylated primary antibody was used, repeat washing step.
  7. (Optional) If biotinylated primary antibody was used, resuspend the cell pellet in buffer and stain with fluorochrome-conjugated Biotin Antibody according to the manufacturer’s recommendations.
  8. Resuspend cell pellet in a suitable amount of PBS for analysis.

Materials

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