Skip to main content

Multicolour flow cytometry analyses and autofluorescence in chlorophytes: lessons from programmed cell death studies in Chlamydomonas reinhardtii


Flow cytometry is a valuable tool in phycological studies. However, endogenous cellular compounds like nicotinamide adenine dinucleotide and chlorophyll a and b autofluoresce, potentially interfering with fluorescent markers. Furthermore, autofluorescent properties are not uniform across algae, nor are their effects consistent in different cytometers. The choice of instrument and fluorescent marker, therefore, requires careful consideration. We investigated the suitability of fluorescent markers by using standard four-colour and advanced multicolour flow cytometers in relation to the effects of autofluorescence over ranges of parameters including fluorophore excitation and emission spectra, band-pass filter configurations, voltage gains and the effects of growth in the light and dark. The unicellular chlorophyte and model organism, Chlamydomonas reinhardtii, was used and findings were correlated with investigations of programmed cell death. As previously found C. reinhardtii autofluoresces in the red, far-red and infrared spectra. This is independent of laser excitation wavelength, and autofluorescence emits and spills over into detection channels of both four-colour and multicolour instruments. Band-pass filter configurations capturing longer wavelength emissions or fluorophores excited or emitted in these longer wavelengths are generally unsuitable. Furthermore, neither dark nor light incubation impacted the autofluorescent signals. Consideration of these algal autofluorescent properties and their spillover effects is required to avoid erroneous results. Recommendations for the use of a range of fluorophores in programmed cell death and other studies in C. reinhardtii using four-colour and multicolour instruments are made.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8


  • Bigos M, Baumgarth N, Jager GC, Herman OC, Nozaki T, Stovel RT, Parks DI, Herzenberg LA (1999) Nine colour eleven parameter immunophenotyping using three laser flow cytometry. Cytometry 36:36–45

    PubMed  Article  CAS  Google Scholar 

  • Chance B (1954) Spectrophotometry of intracellular respiratory pigments. Science 120:767–775

    PubMed  Article  CAS  Google Scholar 

  • Chance B, Cohen P, Jobsis F, Schoener B (1962) Intracellular oxidation-reduction states in vivo. The microfluorimetry of pyridine nucleotide gives continuous measurement of the oxidation state. Science 137:499–508

    PubMed  Article  CAS  Google Scholar 

  • Durand PM, Rashidi A, Michod RE (2011) How an organism dies affects the fitness of its neighbors. Am Nat 177:224–232

    PubMed  Article  Google Scholar 

  • Harris EH (2009) Introduction to Chlamydomonas and its laboratory use, vol 1, 2nd edn, The Chlamydomonas sourcebook. Academic, Amsterdam, p 444

    Google Scholar 

  • Heintzelman DL, Lotan R, Richards-Kortum RR (2000) Characterization of the autofluorescence of polymer monuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia. J Photochem Photobiol 71:327–332

    Article  CAS  Google Scholar 

  • Hulett HR, Bonner WA, Barrett J, Herzenberg LA (1969) Cell sorting: automated separation of mammalian cells as a function of intracellular fluorescence. Science 166:747–749

    PubMed  Article  CAS  Google Scholar 

  • Moharikar S, D’Souza JS, Kulkarni AB, Rao BJ (2006) Apoptotic-like cell death pathway is induced in unicellular chlorophyte Chlamydomonas reinhardtii (Chlorophyceae) cells following UV irradiation: detection and functional analyses. J Phycol 42:423–433

    Article  CAS  Google Scholar 

  • Monici M, Pratesi R, Bernabei PA, Caporale R, Ferrini PR, Croce AC, Balzarini P, Bottiroli G (1995) Natural fluorescence of white blood cells: spectroscopic and imaging study. J Photochem Photobiol 30:29–37

    Article  CAS  Google Scholar 

  • Nedelcu AM (2006) Evidence for p53-like-mediated stress responses in green algae. FEBS Lett 580:3013–3017

    PubMed  Article  CAS  Google Scholar 

  • Nedelcu AM, Driscoll WW, Durand PM, Herron MD, Rashidi A (2011) On the paradigm of altruistic suicide in the unicellular world. Evolution 65:3–20

    PubMed  Article  Google Scholar 

  • Orellana MV, Perry MJ (1995) Optimization of an immunofluorescent assay of the internal enzyme ribulose-1, 5-bisphosphate carboxylase (Rubisco) in single phytoplankton cells. J Phycol 31:785–794

    Article  CAS  Google Scholar 

  • Parks DR, Hardy RR, Herzenberg LA (1984) Three colour immunofluorescence analysis of mouse B lymphocyte subpopulations. Cytometry 5:159–168

    PubMed  Article  CAS  Google Scholar 

  • Prado R, Rioboo C, Herrero C, Suárez-Bregua P, Cid A (2012) Flow cytometric analysis to evaluate physiological alterations in herbicide-exposed Chlamydomonas moewusii cells. Ecotoxicol 21:409–20

    Article  CAS  Google Scholar 

  • Roederer M, De Rosa S, Gerstein R, Anderson M, Bigos M, Stovel R et al (1997) 8 Color, 10 parameter flow cytometry to elucidate complex leukocyte heterogeneity. Cytometry 29:328–339

    PubMed  Article  CAS  Google Scholar 

  • Roederer M, Murphy RF (1986) Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to epidermal growth factor endocytosis by 3 T3 fibroblasts. Cytometry 7:558–565

    PubMed  Article  CAS  Google Scholar 

  • Roederer M (2001) Spectral compensation for flow cytometry: visualisation artifacts, limitations and caveats. Cytometry 45:194–205

    PubMed  Article  CAS  Google Scholar 

  • Sebestyen Z, Nagy P, Horvath G, Vamosi G, Debets R, Gratama JW, Alexander DR, Szollosi J (2002) Long wavelength fluorophores and cell-by-cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual-laser benchtop flow cytometer. Cytometry 48:124–135

    PubMed  Article  CAS  Google Scholar 

  • Segovia M, Haramaty L, Berges JA, Falkowski PG (2003) Cell death in the unicellular chlorophyte Dunaliella tertiolecta. A hypothesis on the evolution of apoptosis in higher plants and metazoans. Plant Physiol 132:99–105

    PubMed  Article  CAS  Google Scholar 

  • Spudich JL, Sager R (1980) Regulation of Chlamydomonas cell cycle by light and dark. J Cell Biology 85:136–145

    Article  CAS  Google Scholar 

  • Zuppini A, Andreoli C, Baldan B (2007) Heat stress: an inducer of programmed cell death in Chlorella saccharophila. Plant Cell Physiol 48:1000–9

    PubMed  Article  CAS  Google Scholar 

Download references


This work was supported by funds from the Medical Research Council, University of the Witwatersrand, and the National Health Laboratory Service (South Africa).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Pierre M. Durand.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kay, P., Choudhury, R., Nel, M. et al. Multicolour flow cytometry analyses and autofluorescence in chlorophytes: lessons from programmed cell death studies in Chlamydomonas reinhardtii . J Appl Phycol 25, 1473–1482 (2013).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Programmed cell death
  • Chlamydomonas reinhardtii
  • Autofluorescence
  • Flow cytometry