TN 230

Technical Note 230

DeNovix Erythrosin B Assay Protocol

Introduction

The Erythrosin B exclusion assay distinguishes between live (unstained) and dead (stained) cells and enables a viability assessment of a cell suspension. Erythrosin B permeates the compromised membranes of dead cells and binds to intracellular proteins, resulting in a dark stained cell.

The Erythrosin B App on CellDrop Automated Cell Counters enables rapid automated cell counting and viability assessment of cell suspensions stained with Erythrosin B.

Kit Contents

Kits include a solution of 0.02% Erythrosin B. The dye should be stored at 4 degrees Celsius in a light-protected tube.

Assay SizeErythrosin B Original ConcentrationNumber of Tests
0.25 mL0.02%50
1.5 mL0.02%300

Sample Volume and Chamber Height

The required sample volume for the CellDrop depends on the height of the measurement chamber, which is set in the counting protocol.

Standard Magnification (FLi & BF)

Gap Height (um)Volume (uL)Minimum Density (cells/mL)Maximum Density (cells/mL)
400407.0E+023.1E+06
100102.9E+031.3E+07
5055.9E+032.5E+07
Higher Magnification (FLxi & BFx)

Gap Height (um)Volume (uL)Minimum Density (cells/mL)Maximum Density (cells/mL)
400404.3E+032.6E+07
100101.7E+041.0E+08
5053.4E+042.1E+08

Best Practices

  • Ensure that the upper and lower chamber surfaces are clean prior to loading sample.
  • Lower the arm prior to dispensing sample into the measurement chamber.
  • Mix cells immediately before loading sample and avoid introducing air bubbles.
  • Follow the image guides to adjust focus and exposure.
  • Allow cells to settle and stop moving across the live preview before pressing the Count button.
Figure 1: Correct focus and exposure settings.

Sample Prep

  1. Vortex cell suspension prior to use.
  2. Mix Erythrosin B and cell suspension together at the desired ratio and mix well. There is no incubation time required. Refer to this table:
Erythrosin B VolumeCell VolumeProtocol Dilution FactorRecommended Exposure
5 µL 0.02%5 µL2Normal

Sample Measurement

  1. With the CellDrop arm in the down position, launch one of the Erythrosin B apps.
  2. Set sample name, information and protocol as appropriate. If mixing cells and Erythrosin B in a ratio other than 1:1, edit the Dilution Factor in the protocol.
  3. Pipette well-mixed cells + Erythrosin B solution and dispense appropriate sample volume 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.
  4. Adjust exposure and focus according to the image guide.
  5. Allow cells to settle, then press the Count button.

Refer to Technical Note 186 – CellDrop Best Practices for additional guidance and Technical Note 226 for a comparison of Erythrosin B with AO/PI and Trypan Blue.

Refer to denovix.com/sds for safety data sheets for CellDrop Cell Counting Assays.

9-OCT-2024