Materials and Reagents

1.5 mL amber microcentrifuge tubes (Abdos, catalog number: P10204A)
3 mL round bottom polystyrene tubes (Becton Dickinson, catalog number: 156758)
20 mL 1 M HEPES pH 8.0 (Himedia, catalog number: RM380-500G)
Glass slides (Borosil, catalog number: BT409100P02)
Cover slips (Borosil, catalog number: 9115S01)
1 mL pipette tips (Abdos, catalog number: P10109)
200 µL pipette tips (Abdos, catalog number: P10140)
10 µL pipette tips (Abdos, catalog number: P10116)
1 cm quartz cuvette (Shimadzu, catalog number:226-85010-92)
0.2 µm size Millipore membrane filter (Merck, catalog number: GSWP04700)
BD FACS Clean Solution (0.1% Sodium hypochlorite) (Becton Dickinson, catalog number: 340345)
Hydrogen Peroxide (Merck, catalog number: 1.93408.0521)
Milli-Q water
Exponentially growing cultures of Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803 and Fremyella diplosiphon BK14 grown in BG11 medium supplemented with 20 mM HEPES (Allen, 1968).
Note: Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803, and Fremyella diplosiphon BK14 (Kehoe and Grossman, 1996) were grown in BG11 liquid medium supplemented with 20 mM HEPES. Cells were inoculated from a solid BG11+20 mM HEPES agar plate. Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803 were grown under PAR (photosynthetically active radiation) (~80 µmol m^-2 s^-1), while Fremyella diplosiphon BK14 was grown in red light (Red LED Fluorescent light, Havells, India) (~20 µmol m^-2 s^-1) with ~150 rpm shaking at 25°C. The growth curves of all organisms were monitored, and cultures were maintained in the exponential phase by regular sub-culturing.
DCFH-DA (Sigma-Aldrich, catalog number: D6883)
Ethanol (Merck, catalog number: 1.00983.0511)
Disodium hydrogen phosphate (Na₂HPO₄) (Merck, catalog number: 1.93622.0521)
Sodium dihydrogen phosphate (NaH₂PO₄) (Merck, catalog number: 1.93624.0521)
Sodium chloride (NaCl) (SRL, catalog number: 41721)
2 mM (w/v) DCFH-DA Stock solution (1 mL) (see Recipes)
0.1 M PBS pH 7.4 (100 mL) (see Recipes)

Equipment

BD FACSCalibur flow cytometer (Becton Dickinson, catalog number: 342975)
UV-VIS Spectrophotometer 1800 (Shimazdu, catalog number: 80626)
Nikon 90i eclipse fluorescence microscope (Nikon, Japan)
Cooling Centrifuge (Remi, India/NEYA16R)

Software

BD CellQuest Pro (Becton Dickinson, USA)
NIS elements AR software 4.0 (Nikon, Japan)

Procedure

Optimization of FCM parameters
1. Take 50 mL exponentially growing cultures of unicellular (S. elongatus PCC 7942 and Synechocystis sp. PCC 6803) and filamentous (Fremyella diplosiphon BK14) cyanobacteria grown under the abovementioned conditions (item 14 of Materials and Reagents).
2. Remove growth medium by centrifugation at 6,000 × g and 25°C for 10 min and dilute the samples with 0.1 M PBS buffer to different O.D.₇₅₀, i.e., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8. Make 1 mL final volume of each cell concentration in a 1.5 mL microcentrifuge tube.
3. Clean nozzle of BD FACSCalibur with 0.1% sodium hypochlorite solution and Milli-Q water.
  Note: Turn on the BD FACSCalibur flow cytometer 10–15 min before conducting the experiment. Calibration of BD FACSCalibur with QC beads at regular intervals is recommended.
4. Draw two dot plots in BD CellQuest Pro software, i.e., Forward Scatter (FSC) vs. Side Scatter (SSC) and FSC vs. Fluorescence channel 3 (FL3). Similarly, draw two histogram plots of Fluorescence channel 1 (FL1) and FL3. Set log scale for all parameters. FL1 and FL3 are two Fluorescence channels that detect DCF fluorescence (green fluorescence, emission range 515–545 nm) and autofluorescence of Chlorophyll (Chl) ɑ (red fluorescence, emission range >670 nm), respectively. The FSC vs. SSC plot provides information about cell size, shape, and granularity; the FSC vs. FL-3 plot is used to select the cyanobacterial population-based on Chl ɑ autofluorescence and cell size. The FL-1 and FL-3 histogram plot provides the fluorescence intensity of DCF and Chl ɑ, respectively.
5. Transfer samples from step 2 to a 3 mL round bottom polystyrene tube and acquire all samples with a low flow rate (12 μL/min), adjusting the PMT voltages of FSC, SSC, FL1, and FL3 for different organisms, as shown in Figure 1A. The adjusted optimal PMT voltage is used for analyzing all the samples of the same organism, which provides fluorescence intensity from the cell in the absence of DCFH-DA. This background fluorescence of cells is excluded while measuring the mean fluorescence intensity of DCF in DCFH-DA treated cells to estimate ROS levels. Set FSC as the primary parameter and FL3 as the secondary parameter.
6. Acquire 50,000–100,000 events. Keep sample acquisition rate below 1,000 events/second to avoid exposure of more than one cell to laser beam at the same time.
7. Note the minimum O.D.₇₅₀ (from step 2) value that gives sample acquisition rate between 500–1,000 events/second to determine the appropriate cell density of each cyanobacterium required for getting proper signals from FCM.
Figure 1. Optimization of different flow cytometry (FCM) parameters for measurement of reactive oxygen species (ROS) levels using 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH-DA). FCM histogram diagram for optimizing PMT voltage of different fluorescent channels of a flow cytometer (A). Graph of optimization of DCFH-DA concentration for test organism (B). Dot plot diagram of gating the population of interest based on FL3 (Chlorophyll ɑ) fluorescence and FSC-H (relative size) (C). Dot plot diagram of FSC-H vs. SSC-H showing relative size and cellular complexity, respectively, of the test organism (D).
Optimization of DCFH-DA dye concentration
1. Take 10 mL sample of optimized O.D.₇₅₀ (refer to steps 2–7 of section A) for each organism in a 15 mL Falcon tube. Add 100 mM H₂O₂to each sample and incubate for 1 h under growth conditions described in item 14 of the Materials and Reagents section.
2. After incubation, pellet cells by centrifugation at 6000 × g and 25°C for 10 min, and wash the cells twice with 10 mL of 0.1 M PBS (pH 7.4).
3. After washing, suspend the cells in 10 mL of 0.1 M PBS and divide into 1 mL of aliquots.
4. Add different concentrations (0, 10, 15, 20, 25, 30, 35, and 40 µM) of DCFH-DA to 1 mL of cell aliquots using DCFH-DA stock solutions (item 20 of Materials and Reagents section).
5. Incubate cells for 1 h in the dark with ~150 rpm rocking at 25°C.
6. Analyze cells using FCM immediately after 1 h of incubation.
7. Wash FCM and set BD CellQuest Pro software with optimized PMT voltage (refer to section A).
8. Acquire samples with low flow rate (12 μL/min).
9. Determine the minimum concentration of dye required for in vivo detection of ROS for each organism by analyzing the titration curve, as shown in Figure 1B.
Detection of in vivo ROS using FCM
1. After determining the optimum cell and dye concentration for each organism (refer to sections A and B), take cyanobacterial cells from different experimental conditions. Here, we used different cyanobacteria that were grown in conditions mentioned in item 14 of the Materials and Reagents section.
2. Pellet cells by centrifugation at 6,000 × g and 25°C for 10 min, and wash the cells twice with 1 mL of 0.1 M PBS (pH 7.4). After washing, resuspend cells in 1 mL of 0.1 M PBS buffer.
3. Incubate cells with the determined concentration of dye for 1 h in the dark with rocking.
4. After incubation, separate samples in two microcentrifuge tubes and immediately acquire one of them with the help of a BD FACSCalibur flow cytometer using optimized settings. Use another aliquot of cells for ROS detection by fluorescence microscopy, as described by Rastogi et al. (2010) (Figure 2).
  
  Figure 2. Measurement of reactive oxygen species (ROS) in Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803, and Fremyella diplosiphon BK14 using 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH-DA) and fluorescence microscopy or Flow cytometry (FCM). Dichlorofluorescein (DCF) fluorescence showing ROS levels (Green) and autofluorescence of photosynthetic pigments (Red) in F. diplosiphon BK14, S. elongatus PCC 7942, and Synechocystis sp. PCC 6803 (A). Fluorescence from DCFH-DA only sample without cells represents the negative control. Images were acquired using a ×40 objective. Scale bars, 5 μm. FCM histograms showing fluorescence of dichlorofluorescein (DCF), i.e., ROS level, in S. elongatus PCC 7942, F. diplosiphon BK14, and Synechocystis sp. PCC 6803 (B). Histograms showing background fluorescence of FL1 channel from S. elongatus PCC 7942, F. diplosiphon BK14, and Synechocystis sp. PCC 6803 without DCFH-DA (C).
5. Acquire at least 50000 events at a low flow rate (12 μL/min).
6. Save the data in FCM format.

Data analysis

The flowchart (Figure 3) shows steps involved in the detection of ROS (steps 1–3; refer to sections A, B, and C), and data analysis.
Using BD CellQuest Pro software, gate cyanobacterial population with the help of FSC vs. FL3 (x-axis FSC and y-axis FL3) (Figure 1C).
Generate histogram diagram of FL1 as shown in Figure 2.
Record the mean fluorescence intensity (MFI) of the FL1 histogram from the gated population of cyanobacterial cells, which depicts the ROS level.
MFI of FL1 channel histogram can also be represented as a bar diagram to depict the ROS level.
The values of FL1 from different organisms can be analyzed using one-way ANOVA with Tukey post hoc function or other suitable statistical analysis.

Figure 3. Steps involved in the detection of reactive oxygen species (ROS) and data analysis.

Notes

To optimize the concentration of DCFH-DA, use a concentration of H₂O₂ that gives maximum ROS in the organism while killing less than 10% of the cells (Mondal et al., 2020). A live-dead assay can be performed by using different concentrations of H₂O₂and the live cell-permeable nucleic acid binding fluorescent dye SYBR Green I. Cells were treated with different concentrations of H₂O_2, and live cells were detected using Sybr Green I and FCM to determine the concentration of H₂O₂ that causes less than 10% killing (Mondal et al., 2020).
Flow cytometers have limitations regarding cell/filament size. The nozzle can analyze approximately 1 µm to 50 µm cell/filament length. Though the size range may vary, it is recommended to be careful when using filamentous cyanobacteria or organisms which make clumps or aggregates. Long filaments may clog the nozzle. It is advised to prepare short filament by using a sonicator before acquisition of sample through FCM, if required. However, impact of sonication on ROS levels and cell survival should be analyzed in advance. Samples can be sonicated in an ultrasonic bath sonicator for different time intervals, and fragmentation can be monitored by light microscopy. Filaments of Fremyella diplosiphon BK14 are shorter (<50 µm), and therefore fragmentation was not required for this cyanobacterium.
DCFH-DA is a light sensitive fluorescence probe. It is advised to perform the entire experiment in minimal light and avoid direct exposure of samples and dye to light.
Alternatively, the cell concentration can be determined by direct cell counting or turbidity measurement of the cell sample as scattering of light. A minimum of 1×10⁵ cells are usually required for the acquisition in FCM.

Recipes

2 mM (w/v) DCFH-DA Stock solution (1 mL)
1. 1 mL of 100% ethanol (Molecular Grade).
2. Dissolve 0.974 mg of DCFH-DA in 1 mL of 100% ethanol.
3. Mix by vortexing and store in the dark at -20°C (Can be stored for up to 3 months).
0.1 M PBS pH 7.4 (100 mL)
1. Mix 40 mL of 0.2 M Disodium hydrogen phosphate (Na₂HPO₄) stock with 10 mL of sodium dihydrogen phosphate (NaH₂PO₄) stock.
2. Add 0.9 g NaCl and stir until dissolved.
3. Bring the volume to 100 mL with distilled H₂O and adjust the pH to 7.4.
4. Filter through 0.2 µm size Millipore membrane filter before use.
5. Store at 4°C.

Acknowledgments

This work was supported by the funding from Institute of Eminence incentive grant, Banaras Hindu University (R/Dev/D/IOE/Incentive/2021-2022/32399) and SERB, New Delhi, India, in the form of Early Career Research Award (ECR/2016/000578) to Shailendra P. Singh. Soumila Mondal is thankful to UGC and BHU for providing UGC Research Fellowship. The author acknowledges DBT Interdisciplinary School of Life Sciences (ISLS) BHU for flow cytometry and fluorescence microscopy facility. This protocol was adapted from the procedure published by Mondal et al. (2020). Careful reading and suggestions from Pankaj K. Maurya and Anjali Gupta is also acknowledged.

Competing interests

The authors declare no competing financial and non-financial interests.

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