Cell surface staining protocol using REAlease™ antibodies

Protocol for immunostaining of cell surface markers for flow cytometric analysis and flow sorting applications. Applicable to REAlease releasable antibodies.

Protocol

Staining with REAlease Antibody

  • The recommended dilution for labeling of cells is 1:50 for up to 106 cells in final volume of 100 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.
  1. Determine cell number.
  2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
  3. Resuspend up to 106 nucleated cells per 98 µL of buffer.
  4. Add 2 µL of the conjugated REAlease Antibody.
  5. 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.
  6. Wash cells by adding 1−2 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
  7. Resuspend cell pellet in a suitable amount of buffer for downstream application.

Removal of REAlease Antibody

  • Volumes given below are for up to 107 stained 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.
  • The recommended incubation temperature is room temperature. Lower temperatures may lead to decreased removal efficiency.
  1. Determine cell number.
  2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
  3. Resuspend up to 107 cells in 980 µL of buffer.
  4. Add 20 µL of REAlease Release Reagent. 
  5. Mix well and incubate for 10 minutes in the dark at room temperature.
  6. Wash cells by adding 1 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely. 
  7. (Optional) For relabeling with REAlease Antibody resuspend the cell pellet in 1970 µL of buffer and add 30 μL of REAlease Stop Reagent. Mix well and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely. Resuspend up to 106 nucleated cells per 68 μL of buffer and add 30 μL of REAlease Stop Reagent. Mix well and proceed with steps 4-6 of the protocol for cell surface staining.
  8. Resuspend cell pellet in a suitable amount of buffer or media for downstream application.

Materials