Exponentially growing Leptospira species were centrifuged at 2,600 g for 15 min, washed three times and resuspended into filter-sterilized spring water (Volvic). All Leptospira species were adjusted to 5x108 leptospires/ml and incubated at room temperature (RT) in the dark. At 21 days, survival was determined by enumeration of colony-forming unit (CFU) on EMJH agar plates. For staining, bacteria were fixed with 4% paraformaldehyde at RT for 15 min, stained with DAPI (1μg/ml, Thermo Fisher) for 10 min and mounted on a slide using Fluoromount mounting medium (Thermo Fisher). Quantification of DAPI staining and roundness was performed using Icy software.
Fluoromount mounting medium
Manufactured by Thermo Fisher Scientific
Sourced in Australia
Fluoromount mounting medium is a water-based, permanent mounting medium designed for preserving and protecting fluorescent-labeled specimens. It is formulated to maintain the integrity and brightness of fluorescent dyes and markers.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Triton 0.1, by Merck Group (3 mentions)
Lysotracker dnd 99, by Thermo Fisher Scientific (2 mentions)
Tcs sp5 confocal system, by Leica (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal system, by Leica (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Gentamicin treatment, by Merck Group (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Stellaris confocal microscope, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Millicell ez slide 8 well, by Merck Group (1 mentions)
9 protocols using fluoromount mounting medium
1
Leptospira Survival Quantification
2
Lysosomal Activity Quantification by Flow Cytometry and Microscopy
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Cells were incubated with LysoTracker DND-99 (100 nM; Thermo Fisher, Waltham, Massachusetts) during 1 hr at 37°C. Cells were then fixed with 4% paraformaldehyde at room temperature (RT) for 1 hr. Fluorescence was analyzed using a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, California). More than 10,000 events per sample were recorded. The analysis was performed using the FlowJo software.
LysoTracker staining was also analyzed using a Leica TCS SP5 Confocal System. Briefly, cells were washed twice with PBS after incubation with LysoTracker DND-99 (1 µM), fixed with 4% paraformaldehyde for 1 hr at RT, stained with DAPI (1 µg/mL, Thermo Fisher) during 10 min mounted on a glass slide using Fluoromount mounting medium (Thermo Fisher). Quantification of LysoTracker staining was performed using Icy software.
LysoTracker staining was also analyzed using a Leica TCS SP5 Confocal System. Briefly, cells were washed twice with PBS after incubation with LysoTracker DND-99 (1 µM), fixed with 4% paraformaldehyde for 1 hr at RT, stained with DAPI (1 µg/mL, Thermo Fisher) during 10 min mounted on a glass slide using Fluoromount mounting medium (Thermo Fisher). Quantification of LysoTracker staining was performed using Icy software.
3
Leptospira Infection of THP-1 Macrophages
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THP-1 (ATCC® TIB-202) cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Sigma) and 2 mM L glutamine (Gibco). For all experiments, THP-1 cells were differentiated/activated into macrophages by a treatment with 50 nM PMA for 2 days following by a 24 hr incubation without PMA. Activated macrophages were inoculated with Leptospira at a multiplicity of infection (MOI) of 100 bacteria-per-cell during 2 hr following by 1 hr of gentamicin treatment (Sigma) at 100 μg/ml. Before and after gentamicin treatment, cells were washed three times with PBS.
For Carboxyfluorescein succinimidyl ester (CFSE) labelling, leptospires were resuspended in PBS with 5 μM CFSE (Sigma-Aldrich) for 30 min at RT and then washed three times in PBS. After infection, cells were incubated with 100 nM LysoTracker DND-99 (Thermo Fisher) for 1 hr at 37°C. THP-1 cells were washed three times with PBS and then fixed with 4% paraformaldehyde at room temperature for 15 min. Nuclei were stained with DAPI (1 μg/ml, Thermo Fisher) during 10 min and mounted on a side using Fluoromount mounting medium (Thermo Fisher). Fluorescence was analyzed using a Leica TCS SP8 Confocal System. Quantification of CFSE-positive cell was performed using Icy software.
For Carboxyfluorescein succinimidyl ester (CFSE) labelling, leptospires were resuspended in PBS with 5 μM CFSE (Sigma-Aldrich) for 30 min at RT and then washed three times in PBS. After infection, cells were incubated with 100 nM LysoTracker DND-99 (Thermo Fisher) for 1 hr at 37°C. THP-1 cells were washed three times with PBS and then fixed with 4% paraformaldehyde at room temperature for 15 min. Nuclei were stained with DAPI (1 μg/ml, Thermo Fisher) during 10 min and mounted on a side using Fluoromount mounting medium (Thermo Fisher). Fluorescence was analyzed using a Leica TCS SP8 Confocal System. Quantification of CFSE-positive cell was performed using Icy software.
4
Immunofluorescence Staining Protocol
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Sections were incubated at 37°C for 20 min, washed 3× in 1× PBS for 10 min each, and blocked in blocking buffer (10% BSA, 6% NDS, 0.3% Triton X-100 in 1× PBS) at room temperature for 1 h. The primary antibody was diluted in blocking buffer and applied to the slide, incubated in a humidified chamber overnight at 4°C. Sections were subsequently washed 3× in PBS-T (0.1% Triton X-100 in 1× PBS) for 10 min, and incubated with secondary antibody diluted in PBS-T for 1 h at room temperature. After incubation, the sections were washed in 1× PBS and mounted with Fluoromount mounting medium (ThermoFisher, catalog #00-4959-52).
5
Fluorescence Imaging of Antibody Staining
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Cells were fixed and permeabilized as above before blocking with 1% BSA in PBS for 1 h. Primary antibody incubations were done overnight at 4°C followed by PBS wash and Alexa Fluor secondary antibody (Life Technologies) incubations, for 1 h at room temperature. Cells were then washed and mounted with Fluoromount mounting medium (Invitrogen, ThermoFisher Scientific) containing DAPI for nuclei staining. Fluorescence was imaged on a Leica Stellaris confocal microscope using 405 nm, 488 nm, and 568 nm laser lines for the appropriate dyes and a 63× oil immersion objective. Quantitation of fluorescence signal intensities along line regions of interest were exported from the LAS X software and graphs prepared in GraphPad Prism 9.
6
iPSC-Cardiomyocyte Immunofluorescence Analysis
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Cultured iPSC-CF on vitronectin coated glasses were fixed in paraformaldehyde 4% and permeabilized with Triton 0.1% (Sigma). The cells were blocked for 1h30 and incubated during 1 h with primary antibodies (Table 3 ) at room temperature, then secondary antibody for 1h30. Subsequently, cells were incubated with Phalloidin (1:400, A12381; Thermo Fisher Scientific) to stain F-actin. After washing steps, cells were marked with DAPI and slides were mounted using fluoromount mounting medium (Invitrogen). Images were taken with a LSM900 Confocal laser scanning microscope using the × 63 oil immersion objective lens. Immunofluorescence quantifications were made on 5–6 images for each condition. Phalloidin and collagen quantification was performed using the mean fluorescence intensity (MFI) on Fiji. Mitochondrial network analysis quantification was performed using MiNA (Mitochondrial Network Analysis) workflow on Fiji, analyzing the ‘’networks’’ and ‘’mitochondrial footprint’’.
7
iPSC-Cardiomyocyte Immunofluorescence Analysis
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Cultured iPSC-CF on vitronectin coated glasses were fixed in paraformaldehyde 4% and permeabilized with Triton 0.1% (Sigma). The cells were blocked for 1h30 and incubated during 1 h with primary antibodies (Table 3 ) at room temperature, then secondary antibody for 1h30. Subsequently, cells were incubated with Phalloidin (1:400, A12381; Thermo Fisher Scientific) to stain F-actin. After washing steps, cells were marked with DAPI and slides were mounted using fluoromount mounting medium (Invitrogen). Images were taken with a LSM900 Confocal laser scanning microscope using the × 63 oil immersion objective lens. Immunofluorescence quantifications were made on 5–6 images for each condition. Phalloidin and collagen quantification was performed using the mean fluorescence intensity (MFI) on Fiji. Mitochondrial network analysis quantification was performed using MiNA (Mitochondrial Network Analysis) workflow on Fiji, analyzing the ‘’networks’’ and ‘’mitochondrial footprint’’.
8
iPSC-Cardiomyocyte Immunofluorescence Analysis
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Cultured iPSC-CF on vitronectin coated glasses were fixed in paraformaldehyde 4% and permeabilized with Triton 0.1% (Sigma). The cells were blocked for 1h30 and incubated during 1 h with primary antibodies (Table 3 ) at room temperature, then secondary antibody for 1h30. Subsequently, cells were incubated with Phalloidin (1:400, A12381; Thermo Fisher Scientific) to stain F-actin. After washing steps, cells were marked with DAPI and slides were mounted using fluoromount mounting medium (Invitrogen). Images were taken with a LSM900 Confocal laser scanning microscope using the × 63 oil immersion objective lens. Immunofluorescence quantifications were made on 5–6 images for each condition. Phalloidin and collagen quantification was performed using the mean fluorescence intensity (MFI) on Fiji. Mitochondrial network analysis quantification was performed using MiNA (Mitochondrial Network Analysis) workflow on Fiji, analyzing the ‘’networks’’ and ‘’mitochondrial footprint’’.
9
ACE2 Expression in SAEC Cells
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Cells were seeded into 8-well chambers (Millicell EZSLIDE 8-well, Merck, North Ryde BC, New South Wales, Australia) at a density of 2 × 104, allowed to reach 70–80% confluence in 24 h, before treating with condensates (0.1%) or CSE (0.5%, 1%) for either 4 h or 24 h. SAECs were seeded into 8-well chambers coated with bovine collagen I. Cells were then fixed and permeabilized in 4% paraformaldehyde/0.1% Triton X-100, blocked with 10% goat serum for 45 min and incubated with primary ACE2 monoclonal antibody (1:100; ab89111, AbCam, Melbourne, Victoria, Australia) overnight at 4 °C. In each experiment, a negative control was achieved through the replacement of the primary antibody with mouse IgG1 (X0931, Dako, Mulgrave, Victoria, Australia). Goat anti-mouse secondary antibody (1:500; Alexa Fluor 488, Invitrogen, Scoresby, Victoria, Australia) was applied for an hour at room temperature before staining nuclei with DAPI (Invitrogen, Scoresby, Victoria, Australia) for a minute and mounted with Fluoromount mounting medium (Invitrogen, Scoresby, Victoria, Australia).
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