HDP1096 Perfecta3D® 96-Well Hanging Drop Plates Protocol

Plate Handling and Spheroid Formation

Figure 1. Hanging Drop Plate assembly:


Caution! Carry the plate assembly by holding the middle tabs on the longer sides of the tray!


  1. Add water or buffer to the reservoirs located on the peripheral rim of the plate and tray (Figure 1), which are divided by baffles into sections. Add 2 mL per plate reservoir section and 1 mL per tray reservoir section.
    Note: if liquid is used to fill the reservoirs, tilting the plate may result in spilling of the liquid and contamination of hanging drops.
    Optional: Prepare 6 mL per plate 0.5-1.0% agarose solution in water or buffer, heat to melt the agarose, and allow the solution to cool to ~50 °C. Add preheated agarose to reservoirs as described above for buffer or water.
  2. Prepare cell suspension to the desired concentration. Each hanging drop holds 30 to 50 μL, so prepare accordingly - e.g. if 5000 cells per 50 μL drop is desired, dilute cell suspension to 100 cells/μL. Exceding 50 μL will lead to drop instability, therefore our suggested starting volume is 40 to 45 μL.
  3. Form hanging drops by pipetting 30 to 50 μL of cell suspension into each well from the top side of the plate (Figure 2). Hanging drops should be formed on and confined to the bottom of the plate.
  4. Put the lid on the plate and place the assembly into a tissue culture incubator. Within hours, individual cells should start to aggregate and eventually form into spheroids. Spheroid formation time varies with cell type. For culture maintenance, please see the media exchange protocol.

Figure 2. Hanging drop formation


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