Technical Note 185

DeNovix Yeast Assay (FDA/PI) Assay Protocol

Introduction

Yeast cell live/dead staining can be done using Fluorescein Diacetate (FDA) and Propidium Iodide (PI). FDA freely moves through the plasma membrane of diverse organisms from bacteria to mammalian cells. The non-fluorescent FDA taken up by cells is converted the into the green fluorescent metabolite fluorescein. The green fluorescence is used as an indicator of live cells. PI enters only nucleated cells with compromised membranes to generate red fluorescence indicating dead cells.

The Yeast app on CellDrop™ Series instruments enables rapid automated cell counting for live/dead yeast cell suspensions.

Kit Contents

Kits are available in a size appropriate for 300 assays and include PI, FDA and Yeast Dilution Buffer. The PI and Buffer should be stored at 2 – 8oC in an airtight container. The FDA should be stored at -20oC in an airtight container. The PI and FDA should be protected from light.

Figure 2: Verify Green Channel Focus. Left - good focus, Right - out of focus.

Best Practices

  • Ensure that both upper and lower sample surfaces are clean, and lower the arm prior to dispensing a sample onto the measurement chamber.
  • Use the correct dilution factor in the protocol settings.
  • Avoid introducing air bubbles.
  • Adjust the focus so that live cells have bright white centers with a sharp black ring, as shown in Figure 1. Verify focus in green channel prior to count (Figure 2)
  • Adjust the exposure in the red and green channels so that cells are neither under or overexposed, as described in the info button dialog in the exposure menu.
Figure 1: Correct focus will show live cells with bright white centers and sharp black rings.

Sample Prep

  1. Equilibrate all solutions to room temperature before use. Vortex cell suspension, FDA, and PI prior to use.
  2. Spin down yeast cells and, resuspend the pellet in yeast dilution buffer.                                               
    • Note: It may be necessary to further dilute cells using the yeast dilution buffer depending on density, 1:100 dilution is recommended.
  3. Combine 18 µL of yeast cell sample with 1 µL of FDA and 1 µL of PI.
  4. Incubate for 15 minutes at room temperature in the dark and proceed to sample measurement.

Sample Measurement

  1. Ensure that the arm is in the down position, and launch the Yeast 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 enter the appropriate cell Dilution Factor.
  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 the 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. Verify focus in green channel prior to count.
  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 denovix.com/msds for safety data sheets for CellDrop Cell Counting Assays.

Revised 19 Oct 2020