Technical Note 183

DeNovix Propidium Iodide Assay Protocol

Introduction

Propidium Iodide (PI) is a red-fluorescent nuclear and chromosomal stain that only enters dead cells. Upon entry, the PI stain binds to DNA by intercalation, resulting in a red fluorescing dead cell. In aqueous solution, the dye has excitation/emission maxima of 493/636 nm. When bound, the excitation/emission maxima shifts to 535/617 nm respectively.

The PI app uses brightfield images to capture live cell counts and red fluorescence images for counting dead cells. The PI Assay and the PI app on CellDrop™ Series instruments enable rapid automated cell counting for cell suspensions.

Kit Contents

Kits include a solution of PI in PBS. The PI reagent should be stored protected from light at 2 – 8oC in an airtight container.

Assay SizeNumber of Tests
0.25 mL50
1.5 mL300

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 cell counting protocol.
  • Avoid introducing air bubbles.
  • Adjust the focus in the brightfield channel so that cells have bright white centers with a sharp black ring as seen in Figure 1.
  • Adjust the exposure in the red fluorescence channel so that fluorescent cells are not under or overexposed, as shown in the info dialog under 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, and vortex before use.
  2. For each sample, mix PI and a cell suspension together in a 50% solution and vortex.
    • Note: A 50% solution is made up of one part PI and one part cell suspension, resulting in a Dilution Factor of 2 for the cell suspension.
  3. Mix sample thoroughly prior to loading onto the CellDrop.

Sample Measurement

  1. Ensure that the arm is in the down position and launch the PI app.
  2. Clean the sample surfaces if there is visible debris in the preview image.
  3. Optional: Enter a sample name and additional sample information.
  4. Select or create a protocol, and set the Dilution Factor to 2 for a 1-to-1 dye/cell suspension mix.
  5. With the arm down, dispense the sample aliquot  into the measurement chamber using the groove on the lower sample surface as an alignment 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.
  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