Fig 1.
Detection of intramacrophagic parasites using IN Cell Investigator Developer Toolbox.
Raw image from (A) DAPI and (B) HCS CellMask™ Deep Red channels acquired with IN Cell Analyzer 2000 microscope. (C) Nuclei of macrophages identified by a pre-defined nuclear segmentation for objects stained with DAPI larger than 50μm2. (D) Cytoplasm of macrophages recognized by HCS CellMask™ Deep Red staining. (E) Parasites are objects stained with DAPI localized inside cytoplasm and with sizes between 0.25–1μm. (F) Final processed image showing macrophage nuclei (blue line), cell boundaries (yellow line) and parasites (green line). Scale bar, 10μm.
Fig 2.
Detection of intramacrophagic parasites using Cell Profiler.
Raw image from (A) DAPI and (B) HCS CellMask™ Deep Red imaging channels acquired with IN Cell Analyzer 2000 microscope. (C) BMDM nuclei identified as DAPI-positive objects with a size range of 18–50 pixels (green line); also depicted is the nuclear expansion applied to avoid false positive detection of parasites in the vicinity of the nuclei (pink line). (D) Macrophages detected by a propagation algorithm of the HCS CellMask™ Deep Red staining which starts in the nucleus (green line) and ends in the BMDM cell boundaries (pink line). (E) Parasites identified as DAPI-staining objects with 4–12 pixels located within the cytoplasm of BMDMs. (F) Final processed image showing BMDM nuclei expansion (light green line), cell boundary (pink line) and parasites (dark green line). Scale bar, 10μm.
Fig 3.
Intracellular L. infantum labeled with anti-parasite antibodies by IFAT and with DAPI.
Leishmania infantum-infected BMDM stained with (A) anti-cTXNPx and anti-mTXNPx and with (B) DAPI (arrows identify intracellular parasites). (C) Merged image showing co-localization of DAPI-stained (blue) parasites with antibody labeling (red). Images were acquired with an IN Cell Analyzer 2000 microscope. Scale bar, 10μm.
Fig 4.
Heterogeneous morphology of BMDM.
(A) HCS CellMask™ Deep Red raw image acquired with an IN Cell Analyzer 2000 microscope (B) Cytoplasm boundary defined by IN Cell Investigator Developer Toolbox (yellow line). Blue lines represents cell nucleus. Scale bar, 10μm.
Fig 5.
IN Cell Investigator Developer Toolbox infectivity parameters depends on the MOI.
Infection rate and the number of intracellular amastigotes by (A) L. infantum, (B) L. amazonensis using different parasite:BMDM ratios (MOI), as detected by IN Cell Investigator Developer Toolbox. Values represent the mean and standard deviation of three independent experiments.
Fig 6.
Comparison of IN Cell Investigator Developer Toolbox and CellProfiler image analysis protocols with manual counting.
BMDMs were infected with L. infantum axenic amastigotes at different MOI. (A) Infection rate. (B) Number of parasites per infected cell obtained with each automated protocol was compared with manual counting. Each dot represents the number of parasites in a single host cell and horizontal lines show the mean. Around 300 cells counted in each MOI condition. Statistical analysis was performed using the One-Way ANOVA. P-values correspond to *p<0.05.
Fig 7.
Correlation between IN Cell Investigator Developer Toolbox and CellProfiler image analysis protocols.
(A) Infection rate, (B) number of parasites per infected cell, and (C) the total number of cells as compared using both image analysis protocol. Each dot represents the result of a single image.
Fig 8.
Anti-Leishmania activity of amphotericin B assessed with IN Cell Investigator Developer Toolbox and CellProfiler.
Dose response curves of (A) L. infantum or (B) L. amazonensis. IN Cell Investigator Developer Toolbox (red line) and CellProfiler (black line). Values represent the mean and standard deviation of at least two independent experiments.