Flow Cytometric Detection of Reactive Oxygen Species

Materials and Reagents

1. Cells to analyze (this protocol has been successfully performed on A549, CL1-0, and IMR-90 cells)
   
2. Dulbecco's Phosphate-buffered saline (DPBS)
   
3. 2’,7’-dichlorofluorescein diacetate (H₂DCFDA) (Life Technologies, Invitrogen^TM, catalog number: D-399 )
   
4. Anhydrous DMF (N,N-dimethylformamide) (Sigma-Aldrich, catalog number: 227056 )
   
5. N-acetyl-L-cysteine (NAC) (Sigma-Aldrich, catalog number: A7250 )
   
6. Hydrogen peroxide (H₂O₂) (Sigma-Aldrich, catalog number: 349887 )
   
7. 5 ml polystyrene BD falcon round-bottom tube with cell strainer cap (BD Biosciences, catalog number: 352235 )
   
8. Sodium Chloride (NaCl)                     
   
9. Potassium Chloride (KCl)                                       
   
10. Potassium Phosphate, monobasic (KH₂PO₄)                                   
    
11. Sodium Phosphate, dibasic (Na₂HPO₄)
    
12. DPBS (see Recipes)
    
13. H₂DCFDA stock (10 mM) (see Recipes)
    
14. NAC stock (1 M) (see Recipes)
    
15. H₂O₂ stock (1 M) (see Recipes)
    

Equipment

1. Flow cytometry
   
2. Water bath
   
3. Centrifuge
   

Procedure

1. Cells were cultured with complete medium in a 6 cm dish at 37 °C and 5% CO₂.
   
2. Cells were treated with NAC or H₂O₂ after reaching 70-90% confluency. ROS scavenger NAC is a precursor to cysteine and glutathione which are strong antioxidants; while H₂O₂ is a compound with an oxygen-oxygen single bond and is known as a strong oxidizer.
   
3. (For negative control) Incubate cells with freshly prepared 5 mM NAC in culture medium for 1 h at 37 °C after three time washes of pre-warmed DPBS.
   
4. (For positive control) Incubate cells with freshly prepared 0.1 mM H₂O₂ from 1 M stock in DPBS for 20 min at 37 °C after three time washes of pre-warmed DPBS.
   
5. Dilute the H₂DCFDA stock solutions into DPBS to make 0.1 μM working solution.
   
6. Cells were harvested by trypsinization.
   
7. Suspend cells in working solution at a density of 1 x 10⁶ cells/ml and incubate at 37 °C for 30 min and protect from light.
   
8. Centrifuge the tubes at 130 x g for 5 min.
   
9. Remove the supernatant and gently resuspend the cells in pre-warmed DPBS.
   
10. Repeat the wash steps 8 and 9 twice.
    
11. Submit samples to flow cytometry for ROS detection using the 488 nm laser for excitation and detected at 535 nm.
    

Analysis

1. Gate on the main cell population.
   
   
   Figure 1. Cells were analyzed according to their size and granularity. The X-axis represents the forward scatter (FSC) parameter which is relative to the size for the cell. The Y-axis shows the side scatter (SSC) parameter which correlates with the components inside the cell. Gate 1 indicates the main population of the cells we analyzed.

2. Show the intensity of H₂DCFDA of cells in gate 1.
   
   
   Figure 2. Histogram of H₂DCFDA. It shows how many cells are at each intensity of H₂DCFDA. The X-axis represents the H₂DCFDA intensity, while the Y-axis indicates the cell counts in corresponding fluorescence intensity.

Recipes

1. DPBS (1 L)
   8 g Sodium Chloride (NaCl)
   0.2 g Potassium Chloride (KCl)
   0.2 g Potassium Phosphate, monobasic (KH₂PO₄)
   1.15 g Sodium Phosphate, dibasic (Na₂HPO₄)
   Adjust to pH = 7.3.
   
2. H₂DCFDA stock (10 mM)
   Dissolve 10 mg in 2.05 ml DMF to make 10 mM stock.
   Aliquot and store at -20 °C.
   Avoid from light and repeated freeze/thaw cycles.
   
3. NAC stock (1 M)
   Dissolve 1 g in 6.128 ml distilled water to make 1 M stock.
   Filter, aliquot and store at -20 °C.
   Avoid from light and repeated freeze/thaw cycles.
   
4. H₂O₂ stock (1 M)
   Dilute 50 μl H₂O₂ from 35% (=11.6M) in 530 μl sterile deionized water to make 1 M stock.
   

References

1. Chang, H. Y., Huang, H. C., Huang, T. C., Yang, P. C., Wang, Y. C. and Juan, H. F. (2012). Ectopic ATP synthase blockade suppresses lung adenocarcinoma growth by activating the unfolded protein response. Cancer Res 72(18): 4696-4706.