RBCs were cultured in RPMI and spiked P. falciparum ring infected erythrocytes to a final hematocrit of 3% and 0.8% infected blood cells. Cultures were then transferred to 96 microwell plates and exposed to increasing concentrations of 3BP, DCA, 2DG, MET, LND, and SIR prepared in media culture. CQ (80 nM) was used as reference drug to achieve parasite growth inhibition values above 90% (>IC90). Each concentration was assayed in triplicate and kept at 37 °C in a 5% CO2, 93% N2, and 2% O2 atmosphere. After 48 h, parasitemia was determined by fluorescence-assisted cell sorting [FACS] (BD LSRFortessa™ cell analyzer, Becton Dickinson), after staining infected RBCs with SYTO 11 (Molecular Probes, Life Technologies). Non-infected RBCs and infected non-treated RBCs were used as controls.
Syto 11
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYTO-11 is a nucleic acid stain used for the detection and quantification of DNA and RNA in a variety of sample types. It exhibits strong fluorescence upon binding to nucleic acids, allowing for the visualization and analysis of cellular content.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Confocal krypton argon and ultraviolet laser system, by Bio-Rad (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lsrfortessa, by BD (1 mentions)
Molecular probe, by Thermo Fisher Scientific (1 mentions)
Dm2500, by Leica (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Olympus (1 mentions)
Bx 15 microscope, by Olympus (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green supermix, by Bio-Rad (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Bcecf am, by Thermo Fisher Scientific (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Tryptone, by BD (1 mentions)
Sp5 microscope, by Leica (1 mentions)
Albumax 1 lipid rich bsa, by Thermo Fisher Scientific (1 mentions)
Sybr green 1 nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
16 protocols using syto 11
1
Antimalarial Agents Screening Assay
2
Visualizing Bacterial DNA in Nematode Guts
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The undigested bacteria DNA in the gut lumen was stained by vital DNA-binding dye SYTO11 (Molecular Probe, Invitrogen) as previously described [21 (link)]. The L3 and L4 stage worms were collected and washed twice with M9 buffer then transferred into a 1.5 ml of tube containing SYTO11 dye (10 μM in M9) at RT for 2 h in the dark. The stained worms were washed twice with M9 and recovered on Escherichia coli OP50 seeded NGM (nematode growth medium) plates at RT for 1 h in the dark. After recovery, the worms were washed twice with M9 and anaesthetized by lavamisole (200 μM), then mounted on to 2% agar pads and observed under a fluorescence microscope (Leica DM2500) with a FITC filter.
3
3D Confocal Microscopy for Quantitative Analysis of hEECs
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As previously described, hEECs are studied using quantitative 3D confocal microscopy of a Bio-Rad system [26 (link)]. In summary, the laser line (9.0 mV) is directed to hEEC and is filtered to prevent photobleaching of the fluorescent dye. The confocal settings were kept unchanged in all the image recordings. The size between the cell sections (16–20 sections/cell depending on the value of the z line) is kept near zero to construct the real image of the cell in 3D. As reported previously, the nucleus is labeled with the fluorescent probe of nucleic acids, syto-11 (Molecular Probes, Eugene, OR, USA) [26 (link)]. Real 3D images are analyzed using an ImageSpace Rix version 6.5 analyzing system. This program allows quantitative 3D images to be obtained by measuring the volume of the cell (expressed in μm3).
4
ER and Nucleus Staining of Aag2 Cells
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Cells were plated on 18 mm × 18 mm coverslips in a 6-well cell culture plate 48 h previously coated with gelatin (0.2%, vol/vol) (62 (link), 86 (link)). To stain the ER, the cell culture medium was aspirated, and cells were washed with PBS once and incubated for 30 min at 26°C with 1 µM live ER-tracker red dye (Molecular Probes) diluted in the appropriate Aag2 cell culture medium. The ER-tracker solution was replaced by a 1/20,000 solution of SYTO-11 (Molecular Probes) DNA dye for 10 min at 26°C diluted in the appropriate Aag2 cell culture medium, washed twice with PBS, and cells were maintained in Aag2 culture medium during the confocal microscopy observations.
5
Quantifying Trypanosoma cruzi Infection
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T. cruzi trypomastigotes were labeled with 5 µM SYTO®11 (binds DNA, Molecular Probes-Invitrogen, Eugene, OR) or 5 µM carboxyfluorescein succinimidyl ester (CFSE, binds amines, Invitrogen) for 20 min at 37oC. THP-1- or BM- derived mφs were infected and incubated with labeled T. cruzi trypomastigotes, as above. Cells were washed, and SYTO®11 or CFSE fluorescence as an indicator of parasite uptake was determined by using an Olympus BX-15 microscope equipped with a digital camera (magnification 40X). Cells infected with CFSE-labeled parasites were also fixed with 2% paraformaldehyde and visualized on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) acquiring 20,000 events. Further analysis was performed by using FlowJo software (ver. 7.6.5, Tree-Star, San Carlo, CA). Mean Fluorescence intensity (MFI) of CSFE positive cells was used as a relative marker of parasites per cell.
Total DNA from normal and infected cells was isolated by using TRIzol reagent (Life Technologies, Grand Island, NY). Total DNA (100 ng) was used as a template in a quantitative PCR (qPCR) on an iCycler thermal cycler with SYBR Green Supermix (Bio-Rad) and oligonucleotide pairs specific for Tc18S ribosomal DNA (Table S1 ). Data were normalized to murine or human GAPDH, and fold change calculated as 2−ΔΔCt, where ΔΔCt represents the Ct (sample) - Ct (control).
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T. cruzi trypomastigotes were labeled with 5 µM SYTO®11 (binds DNA, Molecular Probes-Invitrogen, Eugene, OR) or 5 µM carboxyfluorescein succinimidyl ester (CFSE, binds amines, Invitrogen) for 20 min at 37oC. THP-1- or BM- derived mφs were infected and incubated with labeled T. cruzi trypomastigotes, as above. Cells were washed, and SYTO®11 or CFSE fluorescence as an indicator of parasite uptake was determined by using an Olympus BX-15 microscope equipped with a digital camera (magnification 40X). Cells infected with CFSE-labeled parasites were also fixed with 2% paraformaldehyde and visualized on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) acquiring 20,000 events. Further analysis was performed by using FlowJo software (ver. 7.6.5, Tree-Star, San Carlo, CA). Mean Fluorescence intensity (MFI) of CSFE positive cells was used as a relative marker of parasites per cell.
Total DNA from normal and infected cells was isolated by using TRIzol reagent (Life Technologies, Grand Island, NY). Total DNA (100 ng) was used as a template in a quantitative PCR (qPCR) on an iCycler thermal cycler with SYBR Green Supermix (Bio-Rad) and oligonucleotide pairs specific for Tc18S ribosomal DNA (
6
Cell Staining Fluorescent Probes
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Syto 11, fura-2/AM, BCECF/AM and propidium iodide (all from Molecular Probes—Thermo Fisher Scientific) were dissolved in DMSO, DMSO/20% (w/v) Pluoronic (1:1) or water, respectively at 1 mM concentration. Tryptone was obtained from BD Biosciences. Other reagents and chemicals were obtained from Sigma (St. Louis, MO).
7
3D Confocal Imaging of Cellular Structures
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Cells were examined with a Bio-Rad confocal krypton-argon and ultraviolet laser system, as previously described (Bkaily et al. 1997 (Bkaily et al. , 2004 (Bkaily et al. , 2017;; Ahmarani et al. 2013) .
Briefly, the 488 nm argon laser line (9.0 mV) was directed to the sample via a 510 nm primary dichroic filter and attenuated with a 1%-3% neutral density filter to reduce photobleaching. All the confocal microscope settings (pinhole size, image size, pixel size, laser line intensity, photometric gain, photomultiplier tube settings, and filter attenuation) D r a f t were kept constant throughout the experimental procedures. For each sample, 16-20 sections were recorded covering the entire volume of the cell, constituting a 3dimensional serial section. At the end of each experiment, the nucleus was stained with 100 nmol/L of live cell nucleic acid stain SYTO 11 (Molecular Probes) as described previously (Bkaily et al. 1997 (Bkaily et al. , 2017)) . Scanned images were transferred onto a Silicon Graphics workstation equipped with Molecular Dynamics' ImageSpace analysis and Volume Workbench software modules. The ImageSpace program permits the generation of quantitative 3D images which permits the expression of the measurement per μm 3 .
Images were represented as top-view maximum intensity real 3D projections (not deconvolution) (Bkaily et al. 2017) .
Briefly, the 488 nm argon laser line (9.0 mV) was directed to the sample via a 510 nm primary dichroic filter and attenuated with a 1%-3% neutral density filter to reduce photobleaching. All the confocal microscope settings (pinhole size, image size, pixel size, laser line intensity, photometric gain, photomultiplier tube settings, and filter attenuation) D r a f t were kept constant throughout the experimental procedures. For each sample, 16-20 sections were recorded covering the entire volume of the cell, constituting a 3dimensional serial section. At the end of each experiment, the nucleus was stained with 100 nmol/L of live cell nucleic acid stain SYTO 11 (Molecular Probes) as described previously (Bkaily et al. 1997 (Bkaily et al. , 2017)) . Scanned images were transferred onto a Silicon Graphics workstation equipped with Molecular Dynamics' ImageSpace analysis and Volume Workbench software modules. The ImageSpace program permits the generation of quantitative 3D images which permits the expression of the measurement per μm 3 .
Images were represented as top-view maximum intensity real 3D projections (not deconvolution) (Bkaily et al. 2017) .
8
Confocal Imaging of Nuclear 3D Dynamics
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Cells were examined with a Multiprobe 2001 confocal krypton-argon laser scanning system (Molecular Dynamics, Sunnyvale, California, USA) as previously described (Bkaily et al. 1997 (Bkaily et al. , 2010 (Bkaily et al. , 2017;; Ahmarani et al. 2013) . In brief, all confocal microscope settings (laser line intensity, photometric gain, PMT settings, and filter attenuation) were kept rigorously constant throughout all the experiments (Bkaily et al. 1997 (Bkaily et al. , 1999 (Bkaily et al. , 2010 (Bkaily et al. 2017)) . At the end of each experiment, the nucleus was stained with 100 nmol/L of live cell nucleic acid stain SYTO-11 (Molecular Probes, Eugene, Oregon, USA), as described previously (Bkaily et al. 1997 (Bkaily et al. , 2017)) . Real 3D projections of the nucleus (Bkaily et al. 1997) were used as templates to delineate the nuclear from the cytosolic compartment.
Scanned images were transferred onto a Silicon Graphics workstation equipped with Molecular Dynamics' ImageSpace analysis and Volume Workbench software modules.
For quantification of the 3D images, the ImageSpace program permits the generation of quantitative 3D images which in turn allows fluorescence intensity measurement and expression of the measurement per µm 3 . Images were represented as top-view maximum intensity real 3D projections (not deconvolution) (Bkaily et al. 2017) .
Scanned images were transferred onto a Silicon Graphics workstation equipped with Molecular Dynamics' ImageSpace analysis and Volume Workbench software modules.
For quantification of the 3D images, the ImageSpace program permits the generation of quantitative 3D images which in turn allows fluorescence intensity measurement and expression of the measurement per µm 3 . Images were represented as top-view maximum intensity real 3D projections (not deconvolution) (Bkaily et al. 2017) .
9
Confocal Microscopy and Fluorescence Quantification
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Confocal microscopy, volume rendering and quantitative fluorescence intensity measurement.
Cells were examined with a Multiprobe 2001 confocal krypton-argon laser scanning system (Molecular Dynamics, Sunnyvale, CA) or a Bio-Rad confocal krypton-argon and ultraviolet laser system as previously described (Bkaily et al. 1997 (Bkaily et al. , 2004 (Bkaily et al. , 2017;; Ahmarani et al. 2013) . In brief, laser line intensity, photometric gain, PMT settings, and filter attenuation were kept rigorously constant throughout the experimental procedures (Bkaily et al. 1997 (Bkaily et al. , 1999 (Bkaily et al. , 2017)) . At the end of each experiment, the nucleus was stained with 100 nmol/L of live cell nucleic acid stain SYTO-11 (Molecular Probes, Eugene, OR) as described previously (Bkaily et al. 1997 (Bkaily et al. , 2017)) . Scanned images were transferred onto a Silicon Graphics workstation equipped with Molecular Dynamics' ImageSpace analysis and Volume Workbench software modules. The ImageSpace program permits the generation of quantitative 3D images which permits the expression of the measurement per µm 3 . Images were represented as top-view maximum intensity real 3D projections (not deconvolution) (Bkaily et al. 2017) .
Cells were examined with a Multiprobe 2001 confocal krypton-argon laser scanning system (Molecular Dynamics, Sunnyvale, CA) or a Bio-Rad confocal krypton-argon and ultraviolet laser system as previously described (Bkaily et al. 1997 (Bkaily et al. , 2004 (Bkaily et al. , 2017;; Ahmarani et al. 2013) . In brief, laser line intensity, photometric gain, PMT settings, and filter attenuation were kept rigorously constant throughout the experimental procedures (Bkaily et al. 1997 (Bkaily et al. , 1999 (Bkaily et al. , 2017)) . At the end of each experiment, the nucleus was stained with 100 nmol/L of live cell nucleic acid stain SYTO-11 (Molecular Probes, Eugene, OR) as described previously (Bkaily et al. 1997 (Bkaily et al. , 2017)) . Scanned images were transferred onto a Silicon Graphics workstation equipped with Molecular Dynamics' ImageSpace analysis and Volume Workbench software modules. The ImageSpace program permits the generation of quantitative 3D images which permits the expression of the measurement per µm 3 . Images were represented as top-view maximum intensity real 3D projections (not deconvolution) (Bkaily et al. 2017) .
10
Visualizing Biofilm Structure with Confocal Microscopy
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Confocal laser scanning microscopy (Leica SP5 microscope (Leica, Wetzlar, Germany)) made it possible to visualize the biofilm. Cells were stained with fluorescent dye SYTO® 11 (S7573 ThermoFisher, Waltham, MA, USA) diluted 1:1000 in phosphate buffer. Lectin IV from wheat germ agglutinin (WGA) conjugated with fluorescent dye Alexa Fluor 488 (W11261 ThermoFisher, Waltham, MA, USA) was used for polysaccharide matrix staining [45 (link)]. The Nomarski contrast method allowed for detecting undyed particles. An argon laser with a wavelength of 488 nm (for detecting WGA fluorescence) and 594 nm (for detecting SYTO 11) was used. The resulting images were analyzed using the ImageJ software package with the BioFormats 5.8.2 plugin.
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