Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA)

Materials and Reagents

1. 2 ml RNase, DNase free microcentrifuge tube (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM12425 )
   
2. Glass microscope slides (Fisher Scientific, catalog number: 12-544-4 )
   
3. Glass microscope coverslips (Fisher Scientific, catalog number: S17525B )
   
4. Millipore membrane filter (EMD Millipore, catalog number: HAWP04700 )
   
5. Cuvette (fluorescence spectroscopy) 3 ml (Hellma, catalog number: 101-QS )
   
6. Cyanobacterial cells e.g., Nostoc sp. strain HKAR-2
   Note: Nostoc sp. strain HKAR-2, an autotrophic, filamentous and heterocystous cyanobacterium, was grown under axenic conditions in nitrogen-free liquid BGA medium (Safferman and Morris, 1964) at 20 ± 2 °C under continuous white light (12 ± 2 Wm^-2) to an OD₇₅₀ of 0.8 to 0.9 (exponential growth phase) which was measured using quartz cuvette in a spectrophotometer.
   
7. Nail varnish (Lakme)
   
8. Potassium phosphate dibasic anhydrous (K₂HPO₄)
   
9. Potassium phosphate monobasic (KH₂PO₄)
   
10. 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, catalog number: D6883 )
    
11. 100% ethanol (Sigma-Aldrich, catalog number: 459836 )
    
12. 50 mM phosphate buffer (see Recipes)
    
13. 2 mM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) stock solution (see Recipes)
    

Equipment

1. Glass Petri-dishes (Corning, catalog number: 3160-102 )
   
2. Measuring cylinder
   
3. 295 nm UV cut-off filter (Ultraphan, Digefra, Munich, Germany) to facilitate the desired wavebands of UV-B (280-315 nm), UV-A (315-400 nm) and PAR (400-700 nm)
   
4. UV-treatment chamber fitted with UV-B (Philips Ultraviolet-B TL 40 W: 12, Philips Lighting, model: TL 40W/12 RS SLV/25 ), UV-A (Philips Ultraviolet-A TL 40 W: 12, Philips Lighting, model: TL-K 40W/10-R UV-A ) and PAR (55.08  ±  9.18 μmol m^-2 sec^-1) (OSRAM L 36 W: 32 Lumilux de luxe warm white and Radium NL 36 W: 26 Universal white, Germany) lamps
   
5. Glass rod (Fisher Scientific, catalog number: 11-380A )
   
6. Magnetic stirrer (REMI ELECTROTECHNIK, model: 2 MLH )
   
7. Refrigerated centrifuge (REMI ELECTROTECHNIK, model: CM-12 PLUS )
   
8. Shaker
   
9. Fluorescence microscope (Nikon eclipse Ni fluorescence microscope processed by NIS Elements (BR))
   
10. Fluorescence spectrophotometer (Agilent Technologies, model: Cary Eclipse )
    
11. Spectrophotometer (Hitachi High-Technologies, model: U-2900 , Double beam spectrophotometer)
    
12. Quartz cuvette (3.5 ml) (Cole-Parmer, JENWAY, catalog number: 035 028 )
    

Software

1. NIS-Elements (BR) imaging software (Nikon)
   
2. SigmaPlot 11 software
   

Procedure

A flow chart of the sample preparation is shown in Figure 2.


Figure 2. Flow chart showing various steps involved in the protocol

1. Transfer 100 ml of cyanobacterial culture to sterile glass Petri-dishes with the help of a measuring cylinder. Cover the Petri dishes with a 295 nm UV cut-off filter and transfer them to a UV light treatment chamber.
   
2. Irradiate the cyanobacteria with UV-A, UV-B and PAR and maintain the temperature of the chamber at 25 ± 2 °C to avoid a heating effect. Mix the culture with a glass rod at regular intervals or use magnetic stirrer (15-20 rpm) to avoid self-shading of the cells.
   
3. After desired time intervals (hereafter 12 h and 24 h), take 5 ml of sample and harvest cells by centrifugation at 9,050 x g for 20 min at room temperature.
   
4. Resuspend the cell pellet in 1 ml phosphate buffer (see Recipes).
   
5. Add 2.5 µl of 2 mM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) solubilized in ethanol (see Recipes) to the sample mixture.
   
6. Incubate the sample mixture on a shaker (15 rpm) at room temperature in the dark for 1 h.
   
7. After 1 h incubation
1. For fluorescence microscopy: Take a clean glass slide and add 30 µl of culture. Cover the cells with a glass cover slip. Seal the slide with nail varnish to avoid drying out. Cells were visualized under a fluorescence microscope using an excitation wavelength of 488 nm and emission was detected in the range of 500-600 nm (Figure 3).
   
   
   Figure 3. Fluorescence images of UV-A + UV-B + PAR exposed Nostoc sp. strain HKAR-2 showing green DCF fluorescence after reaction with ROS. Negative control (containing DCFH-DA only; showing the basal level of fluorescence) (A); fluorescence at 0 h (B); 12 h UV-A + UV-B + PAR exposure (C); 24 h UV-A + UV-B + PAR exposure (D). PAR: Photosynthetically active radiation. Scale bar =10 µm.
   
   
2. For fluorescence spectroscopy: 3 ml of liquid sample was added to a cuvette for fluorescence spectrophotometric analysis (Figure 4). The cuvette was placed in fluorescence spectrophotometer and the sample was excited at 485 nm. Emission was recorded in the range of 500-600 nm. The exposure time was limited to 600 msec to reduce the damage of cells. Fluorescence was measured in terms of emitted fluorescence intensity after different durations of stress exposure.
   
   
   Figure 4. Fluorescence intensity of DCF after reaction with ROS generated due to varying duration of UV-A + UV-B + PAR exposure in Nostoc sp. strain HKAR-2 (means ± SD, n = 3). Control: Untreated sample. PAR: Photosynthetically active radiation.
   

Data analysis

The UVR irradiated cells were analyzed using a Nikon Eclipse Ni fluorescence microscope processed by NIS-Elements (BR) imaging software. The microscope was equipped with the following filter set: UV: (DAPI) EX 340 nm EM 488 nm, blue: (FITC) EX 495 nm EM 510 nm and green: (PI 550) EX 550 nm EM 650 nm. Cells were imaged in the epifluorescence mode with a 20x objective lens. The image analysis was performed by NIS-Elements (BR) imaging software provided by Nikon and images were saved in JPEG format. In addition, the fluorescence of the samples was measured by a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies) with an excitation wavelength of 485 nm and an emission band between 500 and 600 nm. The data of the fluorescence spectra were exported to excel and the fluorescence intensity values at 525 nm were extracted. A bar diagram was plotted with SigmaPlot 11 software. All fluorescence measurements were performed at room temperature. All results are presented as mean values of three replicates for fluorescence spectrophotometer analysis and random sites of filaments were used for fluorescence microscopy. All data were analysed by one-way analysis of variance (Brown, 2005). Once a significant difference was detected post hoc multiple comparisons were made by using the Tukey test. The level of significance was set at 0.05 for all tests. All statistical analyses were performed by using SigmaPlot 11 software.

Notes

1. Direct exposure of light to UV treated samples should be avoided.
   
2. The stock solution of 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) once prepared, was kept in -20 °C for further use and direct exposure to light should be avoided (it remains stable for more than 3 months).
   
3. All solutions were filtered through 0.25 µm size Millipore membrane filter before use.
   

Recipes

1. 50 mM phosphate buffer (400 ml)
   8.7 g K₂HPO₄
   6.8 g KH₂PO₄
   Adjust the pH to 7.00
   Filter through 0.25 µm size Millipore membrane filter before use
   
2. 2 mM (w/v) 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) stock solution
   Dissolve 0.974 mg of 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in 1 ml of absolute ethanol
   

Acknowledgments

Rajneesh and Jainendra Pathak are thankful to the Department of Biotechnology (DBT-JRF/13/AL/143/2158) and the Council of Scientific and Industrial Research (09/013/0515/2013-EMR-I), New Delhi, India, respectively, for the financial support in the form of fellowships. SP Singh acknowledges the DST-SERB and UGC for Early Career Research Award and UGC Start-Up Research Grant, respectively. We are also thankful to the Interdisciplinary School of Life Sciences (ISLS), BHU, Varanasi, India, for providing access to the fluorescence microscopy facility. This protocol was adapted from procedures published by Rastogi et al., 2010.

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