Acridine orange staining and quantification by flow cytometry

This is the standard protocol used for the preparation of adherent culture cells used for flow cytometric analysis in this paper.
This information is to serve as a guide and may need to be optimized for particular cell type or flow cytometer equipment.

Sample Preparation

  1. Prepare a 500 ug/mL stock solution of Acridine orange in ddH2O, store at 4oC and protect from direct light. Perform the staining procedures in subdued light;
  2. Grow cells on the desired cell culture plate and induce the cells according to your specific protocol;
  3. Prepare the AO staining solution by diluting the stock solution in pre-warmed culture medium (AO medium) in a concentration of 1.5 ug/mL. Prepare it immediately prior to use;
  4. Harvest the cells by enzymatic release and dissociate cell aggregates;
  5. Add the medium with AO in the proportion 2:1 (AO medium:enzymatic solution) to a final concentration of 1 ug/mL AO. Resuspend the cells by gently pipetting - all samples should be prepared in a single-cell suspension – and transfer to the tube/dish for flow cytometry acquisition;
  6. Incubate the cells for 15 min at room temperature in the dark and proceed with the acquisition on the flow cytometer.


  1. Plot a two-parameter dot-plot of Forward Scatter (FSC) vs Side Scatter (SSC) and continue with standard procedures to gate out debris and doublets;

  2. Plot a two-parameter dot-plot with logarithmic scale to gate out unstained cells. Select the correct bandpass filters, according to the figure 1 of the manuscript, to detect red fluorescence (y-axis) and green fluorescence (x-axis);
    Some software do not permit concurrently selecting two scales (logarithmic and linear) for the same parameter. In this case, skip to the next step. A small percentage of cells may remain unstained.

  3. Plot a two-parameter dot-plot with linear scale to detect red fluorescence (y-axis) and green fluorescence (x-axis);

  4. Introduce the sample and set the machine to “run”. Using the appropriate setting panel, adjust FSC and SSC photo multiplier tube voltages so that the majority of dots in the first two-parameter dot-plot are contained roughly within the center of the plot;

  5. Adjust the red and green fluorescence detectors voltage up or down. The voltage can then be fine-tuned so that in the log scale plot, intensities will be between 102 and 103. This voltage will allow sufficient space along the axes for the most red-stained cells to be held in the graph and not over-spill the end. Fine-tune the voltages so that in the linear scale plot, the events are positioned as represented in the figures along the dot plots represented in the paper;

  6. setting up the machine, collect at least 5.000 gated events.


  1. Percentage of events above a defined threshold

    1. Repeat steps 1-3 from acquisition. In the linear scale dot-plot set an R/GFIRbased threshold (R/GFIR-BT) along the population axis, so that the red-togreen fluorescence ratio is considered, which may be determined by a linear equation 𝑦 = 𝑎x + b, where the slope (a) can be set according to the population distribution.
    2. For the control group, delimit a positive population – above the threshold – from 5 to 10 % and, with the same threshold setting, analyze the other samples. Proportion of cells above selected threshold can be reported as fold change relative to control.

  2. Red-to-green fluorescence intensity ratio (R/GFIR) of individual events

    1. It is possible to analyze each parameter (FSC, SSC, red and green fluorescence intensity) of individual events. These data can be exported, tabulated and analyzed in a suitable presentation package, for example, Excel or Prism. The manner in which these data are extracted may be different for different software.