Modified Kirby-Bauer disc diffusion test (22 (link)) was used to evaluate chronic effect. In brief, 1 × 106 CFUs/ml of each yeast suspension was prepared in phosphate-buffered saline (PBS on YPD supplemented with 0.5 μg/ml methylene blue (MB). Disc filters of 8mm of diameter were impregnated with 0.1, 1, 5, 10, 30 and 50 mM of lugol and then placed on YPD-MB agar plates previously inoculated with 1 × 106 CFU/ml of each strain of Candida evaluated. The plates were incubated at 30°C for 24 h. The presence of inhibition zones or halo (precipitate of methylene blue) was measurement by the diameter of the inhibition zone around each filter using a Motic AE31 inverted microscope. Gap distance of the inhibition zone was measured using Motic Imagine Plus software 2013. To assess the acute effect of Lugol, growth assay was carried out as described previously (23 (link), 24 (link)). All strains were diluted with constant shaking in fresh YPD broth. Each strain was exposed to different concentrations of lugol (0, 0.1, 1, 5, 10, 30, 50 and 100 mM) and after treatment, YPD broth with lugol was removed by centrifugation. The cultures were suspended in distilled water and their OD600 were adjusted to 0.5 and 10-fold. Serial dilutions were made in 96-well plates and 5 μl of each dilution was spotted onto YPD agar plates and photographed in a GelDoc documentation system (BioRad).
Geldoc documentation system
Manufactured by Bio-Rad
Sourced in United States
The GelDoc documentation system is a lab equipment product designed for the visualization and analysis of gel-based samples. It provides a platform for capturing, storing, and analyzing images of electrophoresis gels or other samples. The core function of the GelDoc system is to enable researchers and scientists to document and study the results of their experiments in a reliable and efficient manner.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde, by Merck Group (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Ae31 inverted microscope, by Motic (1 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Quantity one software version 4, by Bio-Rad (1 mentions)
A5502, by Merck Group (1 mentions)
Concentrator plus, by Eppendorf (1 mentions)
7 protocols using geldoc documentation system
1
Evaluation of Lugol's Antifungal Efficacy
2
Evaluation of Macrophage Signaling in Leishmania Infection
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BALB/c-derived peritoneal macrophages were seeded in six-well plates, infected with L. donovani stationary-phase promastigotes, and treated with PPI. Then, the adherent cell populations were collected from the plate and centrifuged at 500 g for 15 min at 4°C. The cell pellet was dissolved in RIPA buffer, and the cell suspensions were sonicated using a probe sonicator. The sample was centrifuged at 14,000 g for 15 min at 4°C, and the protein in samples was estimated using the Bradford method. Then, 50 μg of the total protein was subjected to 10% SDS PAGE gel electrophoresis and transferred to a polyvinylidene (PVDF) membrane. The PVDF membrane was blocked by 5% milk protein in Tris-buffered saline with 0.1% Tween-20 (TBS-T), and immunoblotting was performed to evaluate the levels of protein expressions. The antibody of TLR-4 (SC-293072), p-p38 (SC-166182), pERK (SC-7383), p38 (SC-535), ERK1/2 (SC-514302), and GAPDH (SC-365062) and a secondary antibody of anti-mouse IgG HRP (SIGMA-A9917) served as an internal control. The immunoreactive band was captured by the Bio-Rad Gel Doc documentation system after being visualized with an ECL kit (Bio-Rad), and band intensities were analyzed using ImageJ software (Raja et al., 2017 (link)).
3
Fungal ITS Sequence Analysis and Restriction Profiling
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The ITS sequences from the fungal isolates were first aligned with sequences from NCBI database. The analysis identified two main strains groups, Group A (MAP03, MAP04, and MAP05) and Group B (MAP01, MAP02, and MAP06). Restriction patterns of the ITS sequences were predicted using pDRAW32 and Serial Cloner 1.2. Predicted restriction fragments were compared to choose the best discrimination. Finally, two restriction endonucleases for each group were selected, enzymes Sal I and Alu I for Group A and the enzymes HindIII and MboI for Group B. Digestion was performed by incubating a 5 μL aliquot of PCR product with 10 U of the respective enzyme in a final volume of 20 μL at 37 °C for 2 h. After the electrophoretic separation, which was conducted on 2.5% agarose gels containing ethidium bromide, the images were captured using the GelDoc documentation system (Bio-Rad®, Hercules, CA, USA).
4
Modulating AHCY Expression in Cell Lines
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Stable IMR5/75 and SH-EP cell lines expressing shRNA against AHCY under control of the Tet repressor were generated stepwise as described previously15 (link) using the following oligonucleotide sequence to target AHCY: forward: gatccccGGATCACTACCGCTACTGAttcaagagaTCAGTAGCGGTAGTGATCCtttttggaaa; reverse: gcttttccaaaaaGGATCACTACCGCTACTGAtctcttgaaTCAGTAGCGGTAGTGATCCggg.
IMR5/75 (5,000 cells per well) and SH-EP-AHCYsh (1,000 cells per well) were seeded in 6-well plates and simultaneously treated with Dox (1 µg ml−1) to induce the AHCY-targeting shRNA. Cells were fixed (11% glutaraldehyde; Sigma-Aldrich) and Giemsa-stained five (SH-EP-AHCYsh) or 7 d (IMR5/75-AHCYsh) later. Colony counting was performed using a GelDoc Documentation System and Quantity One software version 4.6.6 (Bio-Rad Laboratories) and quantification using Microsoft Excel 2016.
IMR5/75 (5,000 cells per well) and SH-EP-AHCYsh (1,000 cells per well) were seeded in 6-well plates and simultaneously treated with Dox (1 µg ml−1) to induce the AHCY-targeting shRNA. Cells were fixed (11% glutaraldehyde; Sigma-Aldrich) and Giemsa-stained five (SH-EP-AHCYsh) or 7 d (IMR5/75-AHCYsh) later. Colony counting was performed using a GelDoc Documentation System and Quantity One software version 4.6.6 (Bio-Rad Laboratories) and quantification using Microsoft Excel 2016.
5
Crystal Violet Staining for Colony Counting
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After MMCT, colony of cells were stained with crystal violet for counting. Dishes with cells were washed twice with PBS. Cells were fixed with 4% paraformaldehyde on PBS 15 minutes at RT. After fixation, cells were stained with crystal violet (prepared in 10% ethanol) for 15 minutes at RT. After staining, dishes were gently washed with deionized water until the water no longer runs blue. Images of stained colonies were captured and analyzed using Gel-Doc Documentation System and Quantity One Software (Bio-Rad, Hercules, CA).
6
RAPD Analysis Using Ready-To-Go Kit
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The RAPD analysis undertaken used the Ready-To-Go RAPD kit (GE Healthcare, Bucks, UK) with solely the RAPD Primers 2 (5′-d[GTTTCGCTCC]-3′), 5 (5′-d[AACGCGCAAC]-3′), and 6 (5′-d[CCCGTCAGCA]-3′) employed under the PCR conditions of an initial denaturation for 5 min at 95 °C, followed by 45 cycles at 95 °C for 1 min, 36 °C for 1 min, and 72 °C for 2 min. The final elongation step was increased to 10 min, after which the samples were cooled to 4 °C. The PCR products were analyzed on 2.5% agarose gels containing ethidium bromide, while gel images were captured using the GelDoc documentation system (Bio-Rad®) and the band profile was analyzed for the further generation of phylogenetic trees using the Quantity One 4.6.3 program (BIO-RAD®), scoring 1 for the presence of major bands and 0 for their absence.
7
Inducible Downregulation of AHCY
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IMR5/75-(5,000 cells/well) and SH-EP-AHCYsh (1,000 cells/well) were seeded in six-well plates and simultaneously treated with doxycycline (1 µg/ml) to induce the AHCY-targeting shRNA. Cells were fixed (11% glutaraldehyde; Sigma Aldrich) and Giemsa-stained five (SH-EP-AHCYsh) or seven days (IMR5/75-AHCYsh) later. Colony counting was performed using a Gel Doc Documentation System and Quantity One software (Bio-Rad) and quantification using Excel software. Harvested cells were washed twice with 0.9 % ice-cold NaCl solution and snap-frozen in liquid nitrogen. Frozen pellets were extracted in 180 µl of methanol with vigorous shaking for 15 min at 70°C. As internal standard, 5 µl ribitol (0.2 mg/ml, A5502, Sigma Aldrich) were added to each sample. Polar and organic phases were separated with 100 µl chloroform (shaking samples for 5 min at 37°C), and 200 µl water per sample. Following centrifugation (11,000× g, 10 min), 300 µl of the polar (upper) phase were dried in a vacuum concentrator (Eppendorf Concentrator Plus) without heating for derivatization.
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