Technical Note 181

DeNovix Trypan Blue Assay Protocol


The Trypan Blue exclusion assay distinguishes between live (unstained) and dead (stained) cells. Trypan Blue is a dye that permeates the compromised membranes of dead cells. Upon entry, the dye binds to intracellular proteins, resulting in a dark blue appearance. The Trypan Blue app on CellDrop™ Series instruments enables rapid automated cell counting and viability of cell suspensions stained with Trypan Blue.

Kit Contents

Kits contain 0.4% Trypan Blue in PBS. The Trypan Blue reagent should be stored at room temperature (15 – 30°C) in an airtight container and does not need to be protected from light.

Assay SizeTrypan Original ConcentrationNumber of Tests
0.25 mL0.4%50
1.5 mL0.4%300

Sample Prep

  1. Mix cell suspension and Trypan Blue immediately prior to use.
  2. Optional: Filter Trypan solution through a 0.2 µm filter to remove aggregates and crystals that can form in Trypan solution over time.
  3. For each sample, mix Trypan and a cell suspension together at the desired ratio and vortex. Refer to the table below for Dilution Factor (DF) guidance examples.
Trypan VolumeCell VolumeProtocol Dilution FactorRecommended Exposure
5 µL 0.4%5 µL2Normal
2.5 µL 0.4%7.5 µL1.33Low

Best Practices

  • Ensure that both upper and lower sample surfaces are clean. Lower the arm prior to dispensing a sample onto the measurement chamber.
  • Avoid introducing air bubbles.
  • Once cells are mixed with trypan blue measure within 5 minutes.
  • Adjust the focus and exposure so that unstained cells have bright white centers with a sharp black ring and a sharp transition from light to dark, as shown in Figure 1.
  • Use the correct dilution factor in the protocol settings.
  • Ensure that cells have stopped moving before tapping the Count button.
  • Optimize protocol settings for different cell types. The Default Protocol is a good starting point.
Figure 1: Correct focus and exposure settings.

Sample Measurement

  1. Ensure that the arm is in the down position and launch the Trypan Blue app.
  2. Clean the sample surfaces if there is visible debris in the preview image.
  3. Optional: Enter a sample name and any additional sample information.
  4. Select or create a protocol, and set the Dilution Factor according to the table above.
  5. With the arm down, dispense the sample aliquot into the measurement chamber using the groove on the lower sample surface as a pipetting guide.
    • Note: The volume of sample required depends on the protocol settings for the chamber height. The required volume is displayed on the Count button.
  6. Adjust the focus and exposure.
  7. When the cells have settled and are no longer moving, tap the Count button.

Refer to Technical Note 186 – CellDrop Best Practices for additional guidance.

Refer to for safety data sheets for CellDrop Cell Counting Assays.