Materials and Reagents

Pipette tips 200 μl (Científica Senna, catalog number: 5-20236 )
96-well microplate polypropylene (Thermo Fischer Scientific, Thermo Scientific^TM, catalog number: 267245 )
Glass microscope slide (Corning, catalog number: 2947-75X25 )
Coverslip (Corning, catalog number: 2890-22 )
Note: This product has been discontinued.
Aluminum foil (Reynolds Wrap 15 m x 30 cm)
CellROX^® Green Reagent (Thermo Fischer Scientific, Invitrogen^TM, catalog number: C10444 , Excitation/Emission, 485/520 nm)
Triton X-100 (Karal, catalog number: 9015 )
Methyl jasmonate (abbreviated MeJA) (Sigma-Aldrich, catalog number: 392707-5ML )
Potassium nitrate (KNO₃) (Karal, catalog number: 5082 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Karal, catalog number: 6056 )
Potassium phosphate dibasic (K₂HPO₄) (Karal, catalog number: 5080 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Karal, catalog number: 2016 )
Ethylenediaminetetraacetic acid ferric-sodium salt (Fe·Na·EDTA) (Sigma-Aldrich, catalog number: E6760-100G )
Sulfuric acid (H₂SO₄) (Karal, catalog number: 1032 )
Boric acid (H₃BO₄) (Karal, catalog number: 7021 )
Manganese sulfate monohydrate (MnSO₄·H₂O) (Karal, catalog number: 1069 )
Zinc sulfate monohydrate (ZnSO₄·7H₂O) (Karal, catalog number: 4089 )
Cupric sulfate pentahydrate (CuSO₄·5H₂O) (Karal, catalog number: 8024 )
Sodium molybdate dihydrate (NaMoO₄·2H₂O) (Karal, catalog number: 4072 )
Cobalt(II) sulfate heptahydrate (CoSO₄·7H₂O) (Sigma-Aldrich, catalog number: 12933 )
Note: This product has been discontinued.
Sodium chloride (NaCl) (Karal, catalog number: 6052 )
Potassium chloride (KCl) (Karal, catalog number: 5087 )
Sodium bicarbonate (NaHCO₃) (Karal, catalog number: 5010 )
Sodium phosphate dibasic (Na₂HPO₄) (Karal, catalog number: 6005 )
Modified CHU-13 media (see Recipes)
1x phosphate-buffered saline (PBS) (see Recipes)

Equipment

1.5 L flask (Corning, PYREX^®, catalog number: 4980-1XL )
Micropipettes (Mettler-Toledo International, Rainin^®, catalog numbers: 17014392 , 17014382 and 17011790 )
Incubator shaker (Select BioProducts, model: IncuMix^TM Incubator Shaker, catalog number: SBS256 )
Centrifuge (Labnet International, model: Spectrafuge^TM16M, catalog number: C0160 )
Optical microscope (ZEISS, model: Axio Lab.A1 ) equipped with 470 nm LED module used for fluorochrome excitation and a set of 38 Endow GFP Filters (free exchange (E) EX BP 470/40, BS FT 495, EM BP 525/50) to detect the emission of the fluorochrome
Digital camera (ZEISS, model: AxioCam ICc3 Rev.3 )
pH meter (Cole-Parmer, Jenway, model: 3510 )
Autoclave Sterilmatic (Market Forge Industries, model: STM-EL )

Software

ZEN lite 2011 (ZEISS)
GraphPad Prism version 6.00 for Mac OS X, GraphPad Software, La Jolla California USA (http://www.graphpad.com)

Procedure

Harvest samples of the algal culture grown in modified Chu-13 media (see Recipes), at different time points after specific stress treatments. There should be sufficient colonies in 100 μl to be clearly observed under a microscope. If your alga can be sedimented after centrifugation, 100 μl should make at least a 20 μl of pellet. If the alga doesn’t sediment as in the case of B. braunii, which floats because of the surrounding hydrocarbons, then the OD at 600 nm should give at least 0.5. If needed, concentrate the cells by centrifugation, filtration, or other methods that are suitable for your sample.
Transfer 100 μl of each sample in a 96-well microplate and mix with 2 μl of 5 mM CellROX Green, incubate the microplate for 30 min at 36 °C in the dark by shaking at 120 rpm in an incubator shaker.
Then incline the plates, carefully discard the liquid with a micropipette and wash the cells twice with 100 μl of 1x PBS (see Recipes) containing 0.1% Triton X-100, by shaking at 120 rpm for 5 min at room temperature. Triton X-100 is a commonly used detergent in laboratories widely used to permeabilize the membranes of living cells.
Transfer an aliquot of 10-20 μl of each sample onto a glass microscope slide, cover with a coverslip, and observe under the microscope.
Observe samples first under the white light to locate the algal colonies and then switch to fluorescence conditions. Count at least 100 colonies for each sample.
Colonies with more than 90% of cells having the fluorescent nuclei are considered ROS positive.
Take pictures with a digital camera and the representative images are shown below (Figure 1).

Figure 1. Staining of Botryococcus braunii with CellROX dye. Detect ROS in vivo by analyzing fluorescent nuclei of B. braunii cells in each colony. Upper panels are under white light and lower panels are under fluorescent conditions. A. Control without stress treatment; B. Treatment with strong stress inducer (10 μM MeJA for 60 min); C. Treatment with weak stress inducer (120 mM NaHCO₃, for 60 min).
Determine the percentage of ROS positive colonies according to the equation:

(FC/TC) x 100 = %PC

where, FC = number of colonies with fluorescence, TC = total number of observed colonies, and PC = percent of ROS positive colonies.

Data analysis

With this procedure both the concentration of the inducers and the time of induction are optimized. The number of ROS positive colonies after CellROX staining is determined by counting the fluorescent cells induced with the different treatments for 10, 60 and 120 min. Data represent the mean ± SE of at least three replicates. The statistical significance is evaluated with the Tukey’s test (P value = ** 0.0022, *** 0.0003, **** < 0.0001). Statistical tests are performed using GraphPad Prism version 6.00 for Mac OS X, GraphPad Software, La Jolla California USA (http://www.graphpad.com). Data are visualized using Microsoft Excel for Mac 2011 version 14.1.0. The number of positive colonies for ROS are expressed in % positive colonies (Table 1).

Table 1. Percentage of ROS positive B. braunii colonies. Take samples at different times and with different concentrations of the inducer.

These data show that the best conditions to detect ROS-positive colonies of B. braunii, were 60 min for 10 μM MeJA as well as for 120 mM NaHCO₃. However, the best conditions regarding time and concentration of inducers must be determined for other microalgae.

Notes

A small number of colonies are identified as ROS positive even without treatment of any stress inducer, this may be due to damage during handling of samples. However, they never exceed 13.5% of the total colonies analyzed and are lower than the number of colonies observed in any of the treatments tested.
Due to the colony organization of B. braunii it was sometimes difficult to count individual cells in a selected sample without moving the field under the microscope. So, once the field was fixed under fluorescent light, colonies were considered ROS positive if more than 90% of the cells in the field had fluorescent nuclei. It is recommended to try different focus on the same colony to distinguish the stained nuclei of the cells at different planes.
Triton X-100 was added at the washing steps to improve the introduction of the CellROX Green into the B. braunii cells. In step 2 of the Procedure, when the dye was added, it covered and bonded to the surface of the colony but hardly got into it to act in the inner cells. In step 3, the Triton X-100 allowed the introduction of the dye by its detergent property. This was a key step because of this alga secretes large amounts of liquid hydrocarbons which surround the cells, creating a highly hydrophobic extracellular environment. During the washing steps, the dye reached the inner cells to detect ROS and the excess of CellROX was discarded. In our hands, after the second wash, the reaction stopped because all produced ROS were detected with the dye. Perhaps, other microalgae do not require this step although it may help to get better results, mainly if the cultures were forced to produce a high amount of lipids.

Recipes

Modified CHU-13 media
0.4 g/L KNO₃
0.1 g/L MgSO₄·7H₂O
0.052 g/L K₂HPO₄
0.054 g/L CaCl₂·2H₂O
0.01 g/L Fe·Na·EDTA
5 ml/L trace elements
Dissolve each salt in 800 ml deionized water. Set the pH to 7.2-7.5 using diluted H₂SO₄. The pH of the media should be over 7.0, do not adjust with NaOH. In case the pH is below 7.0, discard the solution and prepare it again
Trace elements:
572 mg/L H₃BO₄
308 mg/L MnSO₄·H₂O
44 mg/L ZnSO₄·7H₂O
16 mg/L CuSO₄·5H₂O
12 mg/L NaMoO₄·2H₂O
18 mg/L CoSO₄·7H₂O
Mix all in 900 ml deionized water in a 1.5 L flask, cover with aluminum foil and sterilize by autoclaving at 121 °C for 20 min
1x phosphate-buffered saline (PBS)
8 g/L NaCl
0.2 g/L KCl
1.44 g/L Na₂HPO₄
0.24 g/L KH₂PO₄
Dissolve the reagents in 800 ml of deionized water. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H₂O to 1 L. Sterilize by autoclaving for 20 min at 121 °C or by filter sterilization. Store PBS at room temperature

Acknowledgments

This technique was adapted from the original procedure described in the Manual of CellROX^® Oxidative Stress Reagents Man0003555 from Life Technologies Corp. This work was supported by a PhD scholarship to IC-C from Consejo Nacional de Ciencia y Tecnología (CONACYT) Mexico, and grant from the 2012 Texas A&M University CONACYT Collaborative Research Grant Program to EL-G and TPD.

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