The amphotericin B solution (250 µg mL−1 in deionized water) was obtained from Sigma-Aldrich (St. Louis, MO, USA), while miconazole nitrate powder was obtained from Sigma-Aldrich (St. Louis, MO, USA) and resuspended in sterile H2O. Caspofungin, fluconazole, 5-fluorocytosine, and butenafine powders were obtained from Cayman Chemical (Ann Arbor, MI, USA) and resuspended in DMSO, ethanol, sterile H2O, and DMSO, respectively. Antifungal drugs were diluted to a working concentration in the 2× RPMI 1640 medium.
5 fluorocytosine
Manufactured by Cayman Chemical
Sourced in United States
5-fluorocytosine is a chemical compound used as a laboratory reagent. It is a fluorinated pyrimidine derivative that can be used in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluconazole, by Cayman Chemical (1 mentions)
Amphotericin b solution, by Merck Group (1 mentions)
Caspofungin, by Cayman Chemical (1 mentions)
Posaconazole, by Cayman Chemical (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Gemini Bio (1 mentions)
Gentamicin sulfate, by Gemini Bio (1 mentions)
Gene pulser, by Bio-Rad (1 mentions)
4 protocols using 5 fluorocytosine
1
Antifungal Drug Preparation Protocol
2
Cell Line Validation and Maintenance
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HC04, Huh7.5.1, and HepG2 cells were obtained from, and initially validated by, ATCC. Subsequently, cells were validated in study by microscopy as all three cell lines, HepG2, Huh7.5.1, and HC04, possess quite distinct morphology. All cell lines were cultured at 37 °C and 5% CO2 in DMEM (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS), 0.29 mg/mL glutamine, 100 units of penicillin, and 100 μg/mL streptomycin. During and after the infection, cell medium was supplemented with antibiotics 50 μg/mL gentamycin (Gemini Bio-Products), 50 μg/mL neomycin trisulfate salt hydrate (SIGMA), 100 units of penicillin (SIGMA) and 100 μg/mL streptomycin (SIGMA), as well as the antimycotics 50 μg/mL 5-fluorocytosine (Cayman) and 100 μg/mL posaconazole (Cayman) were added to the media. Cell lines were tested for mycoplasma contamination prior to being frozen down and new cell aliquots were used for each replicate
3
Hepatocyte Infection with Plasmodium Sporozoites
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Twenty-four hour prior to infection, hepatocytes were either seeded in 24-well (RNA extraction—120,000 per well) or 96-well plates (luciferase growth assays—30,000 per well). Hepatocytes were infected in vitro with P. berghei sporozoites freshly dissected from infected Anopheles stephensi mosquitoes at a ratio of 0.3 sporozoites per seeded cell. Plates were centrifuged at 330 × g for 4 min to bring sporozoites closer to cells, and plates were then incubated at 37 °C 5% CO2 for 2 h to promote sporozoite invasion51 (link). After 2 h, the cells were washed, fresh media containing 12 μM 5-fluorocytosine (Cayman), 50 μg/ml gentamicin sulfate (Gemini Bio-Products), and 100 μg/ml neomycin trisulfate salt hydrate (SIGMA) was added and the cells were returned to the incubator.
4
CRISPR-Mediated Deletion of Pf LINUP
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Marker-free Pf mei2– clone F3 (Goswami et al, 2020 (link)) was used for deletion of Pf LINUP using CRISPR/Cas9 methodology. 5% sorbitol synchronized ring stage Pf mei2– parasites at 5–8% parasitemia were electroporated with 100 µg of pFC-LINUP plasmid at 0.31 kV and 950 µF using a BioRad Gene Pulser (BioRad, Hercules, CA). Positive selection with 8 nM WR99210 (WR; Jacobus Pharmaceuticals, Princeton, NJ) was administered 24 h after the transfection and kept for 5 days. Once drug-resistant parasites were visible on thin blood smears (day 15), parasites were put through two rounds of negative selection pressure with 1 μM 5-fluorocytosine (Cayman Chemical) for 7 days each and cloned by limiting dilution. These parasites were screened by PCR using gene-specific primers (Appendix Table S1 ) and whole genome sequencing to confirm deletion of both Pf Mei2 and LINUP and absence of plasmid DNA.
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