The MNV S7 propagations were produced in RAW 264.7 murine “macrophage-like” cells (ATCC TIB-71) in T75 flasks (Nunc™ EasYFlask™ Cell Culture Flasks, ThermoFisher Scientific) containing approximately 80% confluent cell monolayer according to standard protocols (Gonzalez-Hernandez et al., 2012 (link)). The RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 0.1% (w/v) NaHCO3, HEPES, 10 mM nonessential amino acids, 100 mg/mL penicillin, 100 U/mL streptomycin, 2 mM L-glutamine, and 10% fetal bovine serum (Gibco). Subsequently, the cells were incubated in a 5% CO2−humidified atmosphere at 37°C.
Nunc easyflask cell culture flasks
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc™ EasYFlask™ Cell Culture Flasks are designed for the cultivation and growth of cells in a laboratory setting. These flasks provide a sterile and controlled environment for cell culture operations.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Casein hydrolysate, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 culture medium, by Merck Group (1 mentions)
5 protocols using nunc easyflask cell culture flasks
1
Murine Macrophage-like Cell Culture
2
Cultivation of CSF Amoebae Trophozoites
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CSF samples with trophozoites were cultured in 25 cm2 Nunc™ EasYFlask cell culture flasks (Thermo Fisher Scientific, Waltham, MA, USA). The culture medium consisted of 2% casein hydrolysate (Merck-KGaA, Darmstadt, Germany) in distilled water, supplemented with 10% inactivated fetal bovine serum (Gibco, GranIsland, NY, USA). The flasks were incubated at 37 °C and amoebae were observed daily.
3
Co-culture of PBMC with Cell Lines
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The PBMC were seeded at 2 × 106 – 3 × 106 with the semi-confluent FCK, Hela, and RD cell cultures. The method described by (Graves, Diglio, & Ferrer, 1977 (link)) was followed. In short, after discarding the confluent flask of the FCK cell growth medium, the cells were detached using trypsin/EDTA. The cells were then resuspended, and equal amounts of the adjusted number were mixed with the PBMC in 25 ml Nunc™ EasYFlask™ Cell Culture Flasks (ThermoFisher, USA). The inoculated cultures were incubated at 37 °C and periodically passaged until the cytopathic effects (CPE) were observed.
4
Preparation of Primary Mouse Brain Microglia
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Primary cultures of mouse brain microglia were prepared according to Morales-Ropero et al. (2021) (link). Briefly, newborn (1-day old) C57BL/6 mice were obtained from the animal facility service of the “Centro de Instrumentación Científica” at the University of Granada (UGR) and meninges-free cerebral cortex from the brains were dissected and collected in DMEM with 4.5 g/L D-glucose, 4 mM glutamine, 10% fetal bovine serum, 10% horse serum, 100 U/mL penicillin and 100 μg/mL streptomycin (all reagents from GIBCO, Waltham, Massachusetts, United States). After disaggregation and homogenization, cells were seeded and incubated at 37°C with 5% CO2 for 10–12 days. Then, cultures were softly shaken at 37°C for 2 h and the primary microglia-enriched supernatant was subcultured in the same medium for 2 days before the experiments. Cultures of microglia showed >95% of microglial marker Iba1-positive cells by immunocytochemistry.
BV-2 cells (AcceGen Biotechnology, Fairfield, NJ, United States) were cultured in 25 cm2 Nunc EasYFlask cell culture flasks (Thermo Fisher Scientific, Waltham, Massachusetts, United States) using RPMI-1640 culture medium (Sigma Aldrich, Missouri, United States) supplemented with 10% inactivated fetal bovine serum (Gibco, GranIsland, New York, United States), 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (complete culture medium).
BV-2 cells (AcceGen Biotechnology, Fairfield, NJ, United States) were cultured in 25 cm2 Nunc EasYFlask cell culture flasks (Thermo Fisher Scientific, Waltham, Massachusetts, United States) using RPMI-1640 culture medium (Sigma Aldrich, Missouri, United States) supplemented with 10% inactivated fetal bovine serum (Gibco, GranIsland, New York, United States), 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (complete culture medium).
5
Culturing Naegleria fowleri Trophozoites
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Trophozoites of two clinic isolates of Naegleria fowleri from Costa Rica (accession numbers MT090627 and MT210902) (Retana Moreira et al., 2020b (link)) were cultured in 75 cm2 Nunc EasYFlask cell culture flasks (Thermo Fisher Scientific, Waltham, Massachusetts, United States) with 2% casein hydrolysate (Sigma Aldrich, Missouri, United States) culture medium, supplemented with 10% inactivated fetal bovine serum (Gibco, GranIsland, New York, United States) and antibiotics (penicillin/streptomycin). The flasks were incubated at 37°C, with daily observation of the cultures under an inverted microscope. For each flask, the culture medium was replaced, at least, every 2 days.
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