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. Author manuscript; available in PMC: 2018 Mar 19.
Published in final edited form as: Methods. 2016 Sep 15;112:91–104. doi: 10.1016/j.ymeth.2016.09.007

Table 3.

IFC analysis of intracellular bacterial and protozoan pathogens

Intracellular
parasite		 Fluorescent
reporter		 IFC-
platform		 Assays IFC analysis
(parameters)		 References
Burkholderia thailandensis EGFP Baclighttm Green IS-X Mark II Growth curves; internalization; effect of inhibitor (wortmannin) Max pixel fluorescence; Eroded intracellular and spot count masks [34]
Cryptococcus neoformans Alexa 488; CMDFA IS-X Phagocytosis; stress response; C.neoformans multiplication [50–51]
Brucella melitensis mCherry IS100 Internalization Eroded mask for erythrocytes (BF) from infected mice and 50% intensity mask for mCherry [46]
Leishmania amazonensis CFSE IS-X % infected cells % positive cells; mean number of spots per infected activated cell; fluorescent intensity; max pixel of fluorescence [52]
Leishmania donovani DsRed2 FlowSight Quantitation of infected cells/L.donovani load; NFκB-p65 translocation in infected macrophages MaxPixel vs. intensity, spot counting; similarity score [47]
T.gondii EGFP IS100 Quantitation and detection of T.gondii by size and fluorescence in cell line Spot counting [3]
T.gondii ZsGreenl; mCherry IS-X Quantitation and detection of T.gondii by size and fluorescence in peritoneal excudate cellular subset Multi-color staining and detection T.gondii by size and fluorescence; colocalization; [53]
T.gondii mCherry; tdTomato ZsGreenl IS-X Quantitation and detection of T.gondii by size and fluorescence in peritoneal excudate cellular subset;STAT6 nucleo-cytoplasmic translocation in infected cells Multi-color staining and detection T.gondii by size and fluorescence; colocalization; similarity (DAPI/pSTAT6) [43]
T.gondii mCherry; tdTomato; CellTrace Violet IS-X Quantitation and detection of T.gondii in peritoneal excudate cellular subset Multi-color staining and detection T.gondii by size and fluorescence; colocalization [44]
T.gondii tdTomato IS-X Detection of T.gondii in blood cells 4-color staining of blood cells and detection of T.gondii by size and fluorescence [45]
T.gondii EGFP, EYFP IS** Quantitation and detection of T.gondii in peritoneal excudate subset Multi-color staining and detection T.gondii by size and fluorescence [39]
Salmonella enterica, serovar Thyphimurium EGFP IS-X Internalization; counting bacterial load inside cell Mean number of spots per infected cell; internalization score [39]
Salmonella enterica, serovar Thyphimurium EGFP, AB against O-antigen IS** Bacterial redox “stress” ; intracellular/extracellular bacteria Fluorescent intensities; ratio parameters [54]
Salmonella enterica EGFP IS-X Quantification of Salmonella in infected B-cells Fluorescent intensities [54]
Plasmodium berghei EGFP IS-X Size of merozoits Size [40]
Plasmodium EGFP; RFP IS-X Interaction with erythrocytes Colocalization of EGFP and PKH26 (similarity) [37]
Plasmodium falciparum Fluo-4AM, Hoechst IS100 Phenotypic screen for compounds destroying P.falciparum digestive vacuole Colocalization Fluo-4-AM and DNA dye-stained P. falciparum [56]
Plasmodium falciparum EGFP IS100 Analysis of sorted EGFP-expressing P.falciparum Fluorescent intensity; morphological features [36]
Plasmodium falciparum Hoechst IS** Surface area, sphericity Diameter; perimeter; circularity; projected surface area loss [57]
Plasmodium falciparum Calcein-AM; AnV IS100 Microvesicles (MVs) quantitation by size SSC, bright field and two fluorescent channels to quantitate MVs [58]
Blastocystis spp. CFSE, Hoechst IS-X Mark II Shape, size, granularity, nuclear arrangement Software shape wizard; aspect ratio for CFSE and BF; circularity, granularity; DNA content [59]
Aeromonas veronii* Syto9 (bacteria); AnV; DRAQ5; PI; DHR; IS-X Mark II Phagocytosis; proliferation; apoptosis; NFκB translocation in infected host cells Morphology; similarity score; fluorescent intensity [60]
M.tuberculosis EGFP; Liso Tracker Deep Red IS-X Mark II Phagocytosis;-phagosome maturation; intracellular replication Spot counting; phagosome maturation score [41]
M.tuberculosis AB to CD4, IFNgamma, IL17 IS** Immunophenotyping of MTb-activated dendritic cells Fluorescent Intensities [61]
M.abscessus mCherry; FLICA H2DCFDA IS-X Mark II Apoptotic/necrotic; FLICA/caspase 3/7 and H2DCFDA activated cells Fluorescent Intensities [62]
Entamoeba histolytica PE-anti-TMK antibody IS** Co-localization; phagocytosis Bright detail similarity score (co-localization between fluorescence in different channels) [63]
Entamoeba histolytica No fluorescent reporter IS** Shape Bright field area and circularity score [64]
Entamoeba histolytica CMFDA; DID; live/dead violet dye IS-X Mark II Viability; internalization; amoebic trogocytosis Fluorescent intensities; spot count; Max pixel; internalization score [65]
Pneumocystis carinii PE-CD11c IS-100 Immunophenotyping Fluorescent intensities; Max Pixel values (discriminates punctate from background) [66]
Legionella pneumophila Polyclonal AB to L.p. IS** Detection of L.pneumophila in macrophages Fluorescent intensities; (Legionella-positive punctate) [67]
Francisella tularensis Phalloidin, Fura Red IS** Ca++ production; actin polymerization Fluorescent intensities [68]
Brucella melitensis mCherry IS-100 Detection of mCherry-tagged B.melitensis in red blood cells (free, adherent, overlapping) Fluorescent intensities [69]
Chlamydia pneumoniae Cholera toxin-Al488 IS** Membrane fluidity and uniformity of lipid rafts in C.pneumoniae –infected cells Lipid raft spot counting; homogeneity [70]
*

Aeromonas veronii is an intracellular pathogen of different eukaryotic hosts [71];

IS** model of Imagestream instrument is not specified.