Materials and Reagents

1x Trypsin-EDTA (0.05% / 0.02% in PBS) (PAA Laboratories GmbH, catalog number: L11-004 )
DPBS, Calcium, Magnesium (Life Technologies, Invitrogen^TM, catalog number: 14040 )
HBSS Hank’s Balanced Salt Solution (HBSS) with Calcium and Magnesium and without Red Phenol (Life Technologies, Invitrogen^TM, catalog number: 14025 )
CM-H₂DCFDA (Life Technologies, Invitrogen^TM, catalog number: C6827 )
Propidium Iodide - 1.0 mg/ml (Life Technologies, Invitrogen^TM, catalog number: P3566 )

Equipment

Cell incubator
Six-well plates
Centrifuge
FACSVantage SE (Becton Dickinson)
2 x 75 mm, 5 ml polystyrene round bottom test tube (BD Biosciences, catalog number: 352054 )

Procedure

In the protocol described below, we detect the intracellular ROS after exposure of non cancerous human thyroid epithelial cell line “HTori-3.1” to the oxidative stress. HTori-3.1 cells are grown in complete culture medium (see Recipes).

HTori-3.1 cells were plated at a density of 2.5 x 10⁵ cells per well into six-well plates in complete culture medium.
Sixteen hours later, the cells (90% confluence) were washed by warm DPBS, harvested by trypsinization (250 μl of trypsin / Well).
Add 1 ml of complete culture medium (10% FCS) to stop trypsinization and resuspend cells by gently pipetting up and down.
Collect cells into 5 ml polystyrene round bottom tube by centrifugation (5 min at 200 x g) at room temperature and aspirate supernatant.
Wash cell pellet with 4 ml DPBS and centrifuge 5 min at 200 x g at room temperature.
Resuspend cell pellet in HBSS with 10 μmol/L of CM-H₂DCFDA and increased concentration of H₂O₂ (1 – 5 -10 -15 - 20 - 25 and 50 μmol/L) by gently pipetting up and down (500 μl/tube).
Incubate cells (resuspended in tubes) in cell incubator [(37 °C), high relative humidity (95%), and controlled CO₂ level (5%)] in the dark for 45 min.
Add Propidium Iodide 0.5 μl/0.5 ml/tube (final concentration = 1 μg/ml) and place the tubes on ice (still in the dark) and proceed immediately to flow cytometry analysis. Note that: a) the propidium iodide staining procedure should be performed immediately before analysis on the flow cytometer, b) when we place tubes on ice we minimize cell activity and we avoid artifactual fluctuations of ROS detection between the first tube analyzed in FACS and following tubes.
A total of 5,000 events are analyzed in flow cytometry.
The cellular viability is assessed by propidium iodide staining. Collect PI fluorescence in the FL-3 channel (620 nm) of FACSVantage SE (Becton Dickinson).
Living cells, which are PI negative, were selected by FACS gating. In these living cells, we record the fluorescence of DCF on FL-1 channel (525 nm) of FACSVantage SE (Becton Dickinson).

Analysis

The effect of H₂O₂ concentrations from 1 to 50 μmol/L on the intracellular ROS level was determined by flow cytometry analysis. (Figure 1). We can present results as the " all mean fluorescence "or as "M2 percentage" fluorescence variation. M2 indicate the percentage of cells with an increase production of ROS. We measured a concentration-dependent increase of fluorescence, reflecting an increased level of intracellular ROS (Figure 1).

For example, after exposure of cells to 20 μM H₂O₂, we detect an increase in fluorescence of HTori3.1 cells [Mean fluorescence of all cells is 243.14 in stressed cells and 76.76 in no stressed cells (0 μM H₂O₂) ]. Our results show that the level of ROS in stressed cells is 3 times more than in no stressed cells.
Also, we can analyze results by "M2 and M1 percentage" fluorescence variation. M1 is placed around the no stressed cells. M2 is placed to the right of M1 to designate positive events. We look at the %Gated for M2: 1.23% (no stressed cells) and 47.12% (cells stressed with 20 μM H₂O₂). Our results indicate that 47,12% of cells show an increase in ROS level after exposure to 20 μM H₂O₂.

Recipes

Complete culture medium: phenol red–free RPMI 1640 (Life Technologies, Invitrogen^TM), supplemented with 1% (v/v) antibiotics/antimycotics (Life Technologies, Invitrogen^TM), 2 mmol/L of L-glutamine (Life Technologies, Invitrogen^TM), and 10% (v/v) FCS (PAA Laboratories).

Acknowledgments

This protocol is adapted from Ameziane-El-Hassani et al. (2010).

References

Ameziane-El-Hassani, R., Boufraqech, M., Lagente-Chevallier, O., Weyemi, U., Talbot, M., Metivier, D., Courtin, F., Bidart, J. M., El Mzibri, M., Schlumberger, M. and Dupuy, C. (2010). Role of H₂O₂ in RET/PTC1 chromosomal rearrangement produced by ionizing radiation in human thyroid cells. Cancer Res 70(10): 4123-4132.