Journal of Applied Phycology

, Volume 25, Issue 5, pp 1473–1482 | Cite as

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

  • Patti Kay
  • Rajdeep Choudhury
  • Marietha Nel
  • Monica V. Orellana
  • Pierre M. Durand
Article

Abstract

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.

Keywords

Programmed cell death Chlamydomonas reinhardtii Autofluorescence Flow cytometry 

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Patti Kay
    • 1
  • Rajdeep Choudhury
    • 2
  • Marietha Nel
    • 1
  • Monica V. Orellana
    • 3
  • Pierre M. Durand
    • 2
  1. 1.Department of SurgeryUniversity of the WitwatersrandJohannesburgSouth Africa
  2. 2.Department of Molecular Medicine and HaematologyUniversity of the WitwatersrandJohannesburgSouth Africa
  3. 3.Institute for Systems BiologySeattleUSA

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