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{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;" ! Phagotrophic mixotroph detection (LysoTracker Green) |- | '''Approach:''' LysoTracker Green staining of food vacuoles; comparison to autofluorescence by FCM or imaging FCM |- | '''Context:''' field..."

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{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! Phagotrophic mixotroph detection (LysoTracker Green)
|-
| '''Approach:''' LysoTracker Green staining of food vacuoles; comparison to autofluorescence by FCM or imaging FCM
|-
| '''Context:''' field, lab
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| '''Spatial scale:''' point sample
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| '''Temporal scale:''' hours
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| '''Units:''' active mixotrophic cells L<sup>-1</sup>; % of phytoplankton community
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| '''Community captured:''' phagotrophic mixotrophs
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| '''Co-measurements:''' nutrient uptake measurements; imaging FCM for cell-level detail
|}
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== Method Overview ==

LysoTracker Green (LTG) is an acidotropic fluorescent dye that selectively accumulates in acidic vacuoles within cells. In phagotrophic cells, food vacuoles become acidified after prey ingestion as part of the digestive process, and LTG stains these vacuoles brightly (excitation ~443 nm, emission ~505 nm). When added to seawater samples, cells with active phagosomes/food vacuoles stain green while autofluorescent phytoplankton emit red fluorescence from their plastids. Cells that are simultaneously green (LTG-positive: food vacuoles) and red (plastids) are identified as phagotrophic mixotrophs — organisms that combine photosynthesis with ingestion of prey. The proportion of such dual-fluorescent cells among the phytoplankton community is quantified by flow cytometry or imaging flow cytometry<ref name="Millette2024">Millette, N. C., Leles, S. G., Johnson, M. D., Maloney, A. E., Brownlee, E. F., Cohen, N. R., Duhamel, S., Poulton, N. J., Princiotta, S. D., Stamieszkin, K., Wilken, S., & Moeller, H. V. (2024). Recommendations for advancing mixoplankton research through empirical-model integration. ''Frontiers in Marine Science'', 11. https://doi.org/10.3389/fmars.2024.1392673</ref>.

=== Scale of measurement ===

Discrete samples collected and incubation of hours at in situ temperature required.

=== Data generated ===

Abundance of phagotrophically active mixotrophic cells (cells L<sup>-1</sup>) and their proportion within the total phytoplankton community (%). Imaging FCM additionally provides cell size and morphology data for each counted mixotroph.

=== Units & currency ===

Units are active mixotrophic cells L<sup>-1</sup>. The currency is number of phagotrophic versus phototrophic cells.

=== Sample size ===

Typical samples are ~1 L in volume.

=== Repositories & databases ===

== Limitations ==

Any acidic intracellular compartment — not only food vacuoles — will accumulate LTG, including contractile vacuoles, lysosomes, and chloroplast-associated acidic compartments. This means non-phagotrophic cells may stain green, leading to false-positive identification of mixotrophy. Distinguishing genuine food vacuoles from other acidic compartments requires additional confirmation (e.g., simultaneous FLB ingestion assay or imaging). Heterotrophic protists lacking plastids but with acidic vacuoles could in principle be misidentified as mixotrophs if their autofluorescence is above background.

== Example Applications & Protocols ==

=== Classic examples ===
* Millette et al. (2024) ''Recommendations for advancing mixoplankton research through empirical-model integration'' <ref name="Millette2024" />

=== Recent applications ===
*

=== Common calculations/conversions ===
* % mixotrophic cells = (LTG<sup>+</sup> and autofluorescent cells) / (total autofluorescent cells) × 100.

== References ==

[[Category:Main Pages|Model types]]

LysoTracker Green Incorporation - Revision history