Assay of levels of reactive oxygen species using DHE ratioing

Equipment

Microscopy requirement:

The assay works with wide-field (epifluorescence), confocal and two-photon microscopy.  The epifluorescence microscope has to be capable of low-light level time lapse imaging, e.g. equipped with a fast shutter and a sensitive monochromatic camera. Confocal and two-photon microscopes trivially work in low-light level mode. The image acquisition software must record accurate time stamps for optional rate calculation as 1/s. The assay protocol is given assuming that wide field microscopy and assay automation is used to capture multiple conditions in the same time.

Wide-field microscopy:

• Inverted microscope with 10x or 20x fluorescence-optimized, high NA (0.5-0.75) lens
• Cooled CCD camera
• Fast electronic shutter

Two-photon microscopy:

• 750 nm excitation, low power (e.g. 4% of 1300mW)
• Emission filters specified for wide-field microscopy
• Plate cells in large enough cell culture dish to accommodate a dipping water immersion lens

Imaging medium (IM):

Note: Set pH to 7.4 using NaOH at 37ºC . Store medium aliquoted in 50ml conicals at RT.

Assay protocol (given for LabTek 8-well chamber)

Handling and loading of cell cultures with DHE

1. Culture cells on optical quality plastic bottom or glass bottom plates with equal density when several cell lines are compared. For robustness cells are recommended at 90% confluence on the plates, attached firmly and flattened down. Cell loss is expected due to washes therefore low cell density is not recommended. A confluent culture will prevent accurate background subtraction during data analysis.

2. In a conical warm up sufficient amount of IM (e.g. 20ml for an experiment in a LabTek 8-well  chambered coverglass)

3. Supplement the IM with and sterile filter:

   • zosuquidar  1 µM (optional)

   • BSA 0.4% (can be used with this assay, optionally)

   • glucose 0 to 25mM depending on the experiment - by default match the composition of the culturing media 

   • Note: Zosuquidar is a multi drug resistance protein (MDR) inhibitor  and it may be used for comparison of specimens with different probe accumulation due to pumping or to improve DHE accumulation and increase signal to noise ratio. Note: that sterile filtering is to remove debris from the medium that may interfere with fluorescence imaging.

4. Add 2 µM DHE to 4 ml of supplemented IM (immediately before steps 5 and 6)

5. Wash cell once with supplemented IM

6. Add the DHE containing, supplemented IM to the wells (400ul/well for LabTek 8-well chamber) and incubate cultures for 30 minutes at 37ºC (non-CO₂ incubator). 

7. Incubate cultures at 37ºC for 15 min and start imaging in the presence of DHE.

Imaging

1. Channel setup

   1. DHE OH-ethidium 390nm->617nm

   2. DHE not-yet-oxidized 390nm->460nm

2. Set illumination intensity to see a very grainy, low light level image of the cells, barely distinguishable from the background. Use the 460nm emission to set up view field to stay unbiased for ROS production (the 460nm emission is independent of ROS levels). Use high binning or low scan resolution if possible.

3. Record a time lapse for 20 min with 2 min frame intervals.

4. Alternatively perform treatment and record a new segment of 20min image acquisition.

DHE blue fluorescence in primary pancreatic β-cells	OH-ethidium fluorescence in primary pancreatic β-cells	OH-ethidium fluorescence in primary pancreatic β-cells, masked as the result of the pipeline execution
DHE blue fluorescence intensity graph. The mean of the gray range is calculated	OH-ethidium fluorescence intensity graph.	OH-ethidium rate graph.

Image analysis in Image Analyst MKII

Download tutorial image data set from the Tutorials page.

1. Open the recording ().

2. Activate the "DHE rate of ethidium formation normalized to dihydroethidium" pipeline in the Pipelines/Intensity Measurements/Applications main menu point. Note: this pipeline calculates the rate of increase in OH-ethidium fluorescence intensity (output channel 1) and intensities of the DHE not-yet-oxidized blue fluorescence channel (output channel 2). The image series is registered, background is subtracted, and cell-free areas, determined in a maximum intensity projection image are masked. The output is based on the mean fluorescence intensity of all cells in the view field.

3. Adjust the pipeline parameters:

   1. Channel for...: Make sure that channel numbers listed here match the actual recording

   2. Background Level (percentile): Use a smaller background level (10-30 percentile) for confluent cultures and ~50 percentile for sparse cultures. Percentiles below 5-10 may increase noise.

   3. Mask sensitivity (0.2-1.5): This sets the amount of background masking. At higher values only the brighter parts of the cells are measured, and more background is omitted from the mean fluorescence intensity.

   4. Range for rate measurement: [important] Set the frame range where to measure rates and intensities, ideally from 1 to the last frame (e.g. 1-10).

4. Load the images and run the pipeline by pressing . Note: optionally choose to process the the whole multi-position recording. Note: see a typical result above (right panel).

5. In the Excel Data Window:

   1. For each  position calculate the ratio of Ch1_ROI1 (OH-ethidium rate) and Ch2_ROI1 (DHE blue fluorescence intensity) in an empty column

6. Save results in the Excel Data Window using the File/Save Excel Data Window main menu point.

Protocol by Irina Perevoshchikova and Akos A. Gerencser 11/12/2015 V1.0