Detection and Quantification of Programmed Cell Death in Chlamydomonas reinhardtii: The Example of S-Nitrosoglutathione

Materials and reagents

Biological material

1. We used Chlamydomonas reinhardtii D66 (CC-4425) [8] and CLiP library (CC-4533) [9] strains, but any strain could be used

Reagents

Note: Unless specified otherwise, the reagent can be stored at room temperature.

1. TAP (tris-acetate-phosphate) solution (Life Technologies, catalog number: T8050)

2. S-nitrosoglutathione (GSNO) produced in our laboratory but also available for purchase (Sigma, catalog number: N4148), stored at -20 °C

3. Evans blue powder (Sigma, catalog number: E2129)

4. Phenol:chloroform:isoamyl (25:24:1) (Sigma, catalog number: 77617), stored at 4 °C

5. Ethanol 100% (Sigma, catalog number: 32205-M)

6. Trizma^® base (Sigma, catalog number: 93350)

7. Hydrochloric acid (HCl) (Sigma, catalog number: 258148)

8. Sodium chloride (NaCl) (Sigma, catalog number: S7653)

9. Ethylenediaminetetraacetic acid tetrasodium (EDTA) (Sigma, catalog number: E6511)

10. Sodium dodecyl sulfate (SDS) 20% (Sigma, catalog number: 05030)

11. Sodium acetate (Sigma, catalog number: S2889)

12. RNase A, 10 mg/mL (Thermo Scientific, catalog number: EN0531), stored at -20 °C

13. Agarose (Sigma, catalog number: A9539)

14. Tris borate EDTA (TBE) buffer (Sigma, catalog number: T4415)

15. Ethidium bromide solution (Sigma, catalog number: 46067)

16. GeneRuler 100 bp Plus DNA ladder (Thermo Scientific, catalog number: SM0321), store at 4 °C

17. DAF-FM diacetate (4-amino-5-methylamino-2’,7’-difluorescein diacetate) (Invitrogen, catalog number: D-23844), store at -20 °C, protected from light

1. 50 mM GSNO solution (see Recipes)

2. DNA extraction buffer (see Recipes)

3. Sodium acetate 3.3 M (see Recipes)

4. TBE 0.5× solution (see Recipes)

5. Evans blue solution (see Recipes)

1. 50 mM GSNO solution

   1. Weigh 20 mg of GSNO and dissolve it in 800 µL of TAP medium [10] in a 1.5 mL tube.

   2. Verify the concentration of the 100-fold diluted solution using the molar extinction coefficient of GSNO (922 M^-1·cm^-1 at 335 nm) [11].

   Note: It is better to prepare fresh GSNO for each experiment to avoid its oxidation.




2. DNA extraction buffer (for 500 mL)

   Reagent	Final concentration	Amount
   Tris-HCl (1 M, pH 7.5)	200 mM	100 mL
   NaCl (1 M)	250 mM	125 mL
   EDTA (0.5 M, pH 8)	25 mM	25 mL
   SDS (20%)	0.5%	12.5 mL
   MilliQ water	n/a	262.5 mL
   Total	n/a	500 mL

   Note: It is recommended to autoclave extraction buffer (add SDS after autoclaving) before proceeding to DNA extraction.




3. Sodium acetate 3.3 M (for 100 mL)

   Reagent	Final concentration	Amount
   Sodium acetate	3.3 M	27 g
   MilliQ water	n/a	100 mL
   Total	n/a	100 mL

   1. Dissolve using a magnetic stirrer and stir bar.

   2. Filter sterilize (0.22 µm).




4. TBE 0.5× solution

   Reagent	Final concentration	Amount
   TBE (10×)	0.5×	100 mL
   MilliQ water	n/a	1.9 L
   Total	n/a	2 L




5. Evans blue solution

   Reagent	Final concentration	Amount
   Evans blue powder	0.5 %	0.5 g
   TAP	n/a	100 mL
   Total	n/a	100 mL

   Filter sterilize (0.22 µm).

Laboratory supplies

1. Spectrophotometer semi-micro cuvette (Biosigma, catalog number: BSA002)

2. 24-well plates (Evergreen Labware, Medical Caplugs, catalog number: 222-8044-01F)

3. Surgical tape (Micropore^TM, catalog number: 1530-0)

4. Counting chamber Neubauer (Blaubrand, catalog number: BR717805)

Equipment

1. Spectrophotometer (Implen Nanophotometer)

2. Microplate reader (ClarioStar Plus, BMG LABTECH)

3. Orbitron Rotary shaker (Infors)

4. Olympus BX43 microscope

5. Thermomixer (Eppendorf, catalog number: 5382000015)

6. Vortex (Dutsher Vortex Genie 2, catalog number: 079008)

7. Electrophoresis tanks (Embitec^® runOne^TM, model: EP-2000)

8. Camera (Q-IMAGING Micropublisher 3.3 RTV)

9. NanoDrop One (Thermo Scientific)

10. GelDoc Go Imaging System (Bio-Rad)

Software and datasets

1. Image Lab v6.1 for the GelDoc Go Imaging System (Bio-Rad)

2. Smart control data analysis for ClarioStar Plus (MARS, BMG LabTech)

Procedure

1. Cell culture

   1. Cultivate Chlamydomonas cells in TAP liquid media at 25 °C, under continuous light (50 µmol photons/m/s) with shaking at 120 rpm until you obtain a concentration of 4 × 10⁶ to 6 × 10⁶ cell/mL.

   2. Transfer 1 mL of culture to as many wells as required in a 24-well plate.

   3. Add 20 µL of a 50 mM fresh solution of GSNO per well.

   4. Close the 24-well plate with Micropore surgical tape and place it at 25 °C under continuous light (50 µmol photons/m/s) with shaking at 120 rpm.




2. NO detection after GSNO treatment

   1. Add the DAF-FM diacetate to your sample at a final concentration of 5 µM (e.g., 1 µL of a 5 mM solution in a final volume of 1 mL) and place the cells under low light (e.g., 10 µmol photons/m/s) at 25 °C under agitation (120 rpm) for 30 min.

   2. Centrifuge cells at 2,300× g for 3 min, remove the supernatant, and replace it with the same volume of TAP media. Repeat twice.

   3. After a 15 min incubation period, add GSNO to your sample at a final concentration of 1 mM (e.g., 20 µL of a 50 mM solution in a final volume of 1 mL).

   4. NO can be detected as soon as 30 min after GSNO treatment in a plate reader, using wavelengths corresponding to fluorescein (excitation 483 ± 14 nm and emission at 530 ± 30 nm) (Figure 1).

      
      

      
      Figure 1. Example of nitric oxide (NO) quantification after S-nitrosoglutathione (GSNO) treatment. DAF-FM fluorescence was measured every 15 min for 4 h in a sample treated with GSNO (1 mM) and in the untreated control. Values represent the average of four biological replicates; error bars indicate ± SEM.

      
      

3. DNA degradation profile analysis

   1. Eight hours after the beginning of the GSNO treatment, transfer the contents of two wells (2 mL) of the 24-well plate into a 2 mL Eppendorf tube.

   2. Harvest the cells by centrifuging the tubes at 2,300× g for 5 min.

   3. Remove the supernatant and dissolve the pellet in 600 µL of extraction buffer by placing it for 10 min at 37 °C under 1,400 rpm agitation in a thermomixer.

   4. Centrifuge at 17,000× g for 3 min to pellet the debris.

   5. Transfer 400 µL of the supernatant into a new 1.5 mL Eppendorf tube.

   6. Add 500 µL of phenol:chloroform:isoamyl solution.

   7. Mix the solution by vortexing for at least 30 s.

   8. Centrifuge at 16,000× g for 5 min.

   9. Carefully collect 300 µL of the upper phase and transfer it to a new 1.5 mL Eppendorf tube.

   10. Add 750 µL of 100% ethanol and 45 µL of sodium acetate 3.3 M, mix gently turning the tube over a few times, and leave in ice for 30 min.

   11. Centrifuge at 17,000× g for 30 min at 4 °C to pellet the DNA.

   12. Remove all supernatant and rinse the pellet with 500 µL of ethanol 70%. Centrifuge at 17,000× g for 5 min at 4 °C.

   13. Remove all supernatant and dry the pellet by placing the tube upside down on absorbent paper for 10 min.

       Note: No residues of ethanol should remain at this stage. If needed, the tubes can be dried for a longer time.

   14. Add 100 µL of water and dissolve the pellet by shaking at 1,400 rpm for 5 min at 50 °C in a thermomixer.

       Note: The pellet can be hard to dissolve. If needed, use a pipette tip and increase agitation time.

   15. DNA concentration (typically 200–500 ng/µL) and A_260nm/A_280nm ratio (expressing protein contamination, typically between 1.8 and 2) are estimated spectrophotometrically using a NanoDrop.

   16. Digest 10 µg of DNA with 1 µL of RNase A for 15 min at 37 °C.

   17. Load 10 µg of your DNA samples and 4.5 µL of GeneRuler 100 bp Plus DNA ladder on a 2% agarose gel.

   18. Check the DNA degradation profile after 20 min of migration at 100V and estimate the size of the DNA fragments, using GelDoc Go and Image Lab (Figure 2).

       
       

       
       Figure 2. Analysis of DNA degradation profile in untreated cells compared with cells treated with S-nitrosoglutathione (GSNO) 0.5 mM and 1 mM, 8 h after treatment. For each condition, two independent samples are shown. The size of the marker bands is expressed in base pairs.




4. Death quantification procedure

   1. At different times after GSNO treatment (we recommend 4, 8, and 24 h), take 20 µL of culture from each well and add 8 µL of Evans blue 0.5%.

   2. Observe the cells under a microscope (at 200× or 400× magnification) using a slide with sufficient space to avoid crushing the cells (e.g., Counting chamber Neubauer) and take a representative photo of each sample.

   3. Determine the percentage of dead cells over a population of at least 100 cells by counting the number of blue (dead cells) and living cells (green cells) (Figure 3A). The percentage of dead cells is equal to the number of dead cells divided by the total number of cells, multiplied by 100 (Figure 3B).

      
      

      
      Figure 3. Cell death quantification using Evans blue staining. A. Living cells remain green, while dead cells appear blue after treatment with Evans blue. Scale bar represents 50 µm. B. Percentage of dead cells at different times after S-nitrosoglutathione (GSNO) treatment compared with control. Values represent the average of six biological replicates; error bars indicate ± SEM. Adapted from Lambert et al. [12].

Validation of protocol

This protocol or parts of it has been used and validated in the following research article:

1. Lambert et al. [12]. Type II metacaspase mediates light-dependent programmed cell death in Chlamydomonas reinhardtii. Plant Physiology (Figures 1, 2–6).

   In this article, the percentage of death was assessed in different Chlamydomonas populations, using between four and six biological replicates and a Student's t-test to highlight significant differences. In this way, we were able to reveal quite fine statistical differences between the different samples tested (e.g., Figure 1A).

Acknowledgments

This work was supported in part by the CNRS (MITI, ADAPT-VIVANT), Sorbonne Université (iBio initiative). We would like to thank the authors of the publication from which this Bio-protocol article was inspired: Lambert, L., de Carpentier, F., André, P., Marchand, C. H. and Danon, A. (2024). Type II metacaspase mediates light-dependent programmed cell death in Chlamydomonas reinhardtii. Plant Physiol. 194(4): 2648–2662 [12].

Competing interests

The authors declare no conflicts of interest.

References

1. Kerr, J. F., Wyllie, A. H. and Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 26(4): 239–257.
2. de Carpentier, F., Lemaire, S. D. and Danon, A. (2019). When Unity Is Strength: The Strategies Used by Chlamydomonas to Survive Environmental Stresses. Cells. 8(11): 1307.
3. Lambert, L., de Carpentier, F., André, P., Marchand, C. H. and Danon, A. (2024). Type II metacaspase mediates light-dependent programmed cell death in Chlamydomonas reinhardtii. Plant Physiol. 194(4): 2648–2662.
4. Chang, H. L., Hsu, Y. T., Kang, C. Y. and Lee, T. M. (2013). Nitric Oxide Down-Regulation of Carotenoid Synthesis and PSII Activity in Relation to Very High Light-Induced Singlet Oxygen Production and Oxidative Stress in Chlamydomonas reinhardtii. Plant Cell Physiol. 54(8): 1296–1315.
5. Yordanova, Z. P., Woltering, E. J., Kapchina-Toteva, V. M. and Iakimova, E. T. (2013). Mastoparan-induced programmed cell death in the unicellular alga Chlamydomonas reinhardtii. Ann Bot. 111(2): 191–205.
6. Danon, A., Delorme, V., Mailhac, N. and Gallois, P. (2000). Plant programmed cell death: A common way to die. Plant Physiol Biochem. 38(9): 647–655.
7. Vavilala, S. L., Gawde, K. K., Sinha, M. and D’Souza, J. S. (2015). Programmed cell death is induced by hydrogen peroxide but not by excessive ionic stress of sodium chloride in the unicellular green alga Chlamydomonas reinhardtii. Eur J Phycol. 50(4): 422–438.
8. Schnell, R. A. and Lefebvre, P. A. (1993). Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging. Genetics. 134(3): 737–747.
9. Li, X., Patena, W., Fauser, F., Jinkerson, R. E., Saroussi, S., Meyer, M. T., Ivanova, N., Robertson, J. M., Yue, R., Zhang, R., et al. (2019). A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis. Nat Genet. 51(4): 627–635.
10. Gorman, D. S. and Levine, R. P. (1965). Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci USA. 54(6): 1665–1669.
11. Broniowska, K. A., Diers, A. R. and Hogg, N. (2013). S-Nitrosoglutathione. Biochim Biophys Acta BBA - Gen Subj. 1830(5): 3173–3181.