For C. auris and C. auris fluconazole-resistant mutants (M1 and M2), the viability of the yeasts in the presence of different amounts of fluconazole was determined according to the “Clinical and Laboratory Standards Institute” (CLSI) guidelines M27-A3 broth microdilution assay [47 ]. In brief, 2.5 × 103 yeast cells were seeded in 200 μL of RPMI-1640 medium supplemented with L-glutamine in microtiter plates (flat-bottomed, 96-well, polystyrene) (Sarstedt AG & Co., KG, Nümbrecht, Germany) and incubated at 37 °C for 24 h with agitation at 900 rpm on an Eppendorf shaker. The cell viability was quantified by a resazurin assay, according to Patricia Bi Fai et al. [67 (link)]. The cells were incubated with 20 μL of resazurin at a concentration of 0.15 mg/mL for 2 h. Living cells reduce resazurin to fluorescent resorufin through the production of NADPH. The amount of produced resorufin was analyzed by fluorescence measurements at an excitation wavelength of 535 nm and an emission wavelength of 595 nm with a Tecan Infinite F200 microplate reader to quantify the viability.
Rpmi 1640 medium
Manufactured by Sarstedt
Sourced in Germany
RPMI 1640 medium is a widely used cell culture medium commonly employed for the cultivation of a variety of cell types, including human and animal cells. It provides a balanced salt solution and essential nutrients to support cell growth and maintenance in an in vitro environment.
Automatically generated - may contain errors
5 protocols using rpmi 1640 medium
1
Antifungal Susceptibility of C. auris
2
Gamma Irradiation of Human T Cells
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7.5 × 105 human T cells were transferred in RPMI 1640 medium into a 2 mL reaction tube (Sarstedt, Nümbrecht, Germany). Each reaction tube was treated with a single dose in the range from 2 to 50 Gy as indicated in the respective Figures using the Gammacell® 3000 Elan (Nordion International Inc., Ottawa, ON, Canada).
3
Isolation and Differentiation of Human Macrophages
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Human peripheral blood mononuclear cells were isolated from commercially obtained buffy coats of anonymous donors (DRK-Blutspendedienst Baden-Württemberg-Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Frankfurt, Germany) using Ficoll density centrifugation. Peripheral blood mononuclear cells were washed twice with PBS containing 2 mM EDTA and subsequently incubated for 1.5 h under growth conditions in RPMI 1640 medium supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) to allow their adherence to culture dishes (Sarstedt, Nümbrecht, Germany). Non-adherent cells were removed. Monocytes were then differentiated into naïve macrophages with RPMI 1640 medium (Gibco, Dreieich, Germany) containing 3% AB-positive human serum (DRK-Blutspendedienst Baden-Württemberg-Hessen, Frankfurt, Germany) (complete media) for at least 7 days.
4
Isolation and Differentiation of Human Monocyte-derived Macrophages
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Human monocytes were isolated from commercially obtained buffy coats from anonymous donors (DRK-Blutspendedienst Baden-Württemberg-Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Frankfurt, Germany) using Ficoll density centrifugation. Peripheral blood mononuclear cells were washed twice with PBS containing 2 mM EDTA and subsequently incubated for 1 h at 37°C in RPMI 1640 medium supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml) to allow their adherence to culture dishes (Sarstedt, Nümbrecht, Germany). Nonadherent cells were removed. Monocytes were then differentiated into naïve macrophages with RPMI 1640 medium containing 5% AB-positive human serum (DRK-Blutspendedienst Baden-Württemberg-Hessen, Frankfurt, Germany) for 7 days.
5
Isolation and Differentiation of Human Monocyte-Derived Macrophages
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Human monocytes were isolated from commercially obtained buffy coats from anonymous donors (DRK-Blutspendedienst Baden-Württemberg-Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Frankfurt, Germany) using Ficoll density centrifugation. Peripheral blood mononuclear cells were washed twice with PBS containing 2 mM EDTA and subsequently incubated for 1 h at 37 °C in RPMI 1640 medium supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) to allow their adherence to culture dishes (Sarstedt, Nümbrecht, Germany). Nonadherent cells were removed. Monocytes were then differentiated into naive macrophages with RPMI 1640 medium containing 5% AB-positive human serum (DRK-Blutspendedienst Baden-Württemberg-Hessen, Frankfurt, Germany) for 7 days. Naive macrophages were polarized to AAMs with IL-4 (5 ng/ml) or IL-13 (10 ng/ml), and primed with T0901317 (1 µM) unless indicated otherwise.
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