Saccharomyces cerevisiae yeast (BY4741 strain: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was grown on standard synthetic complete medium (SDC), which consisted of synthetic minimal medium (SD; 0.17% yeast nitrogen base without amino acids, 2% glucose and supplements according to the requirements of the strains) with 0.079% complete supplement mixture (ForMedium, Norwich, UK). Yeast cultures were incubated at 30°C, and growth of cells untreated or treated with edelfosine was monitored by optical density at a wavelength of 595 nm (OD595). Cells were incubated for the indicated times and sample aliquots were taken to measure absorbance at 595 nm. Edelfosine was used at the concentrations indicated in the corresponding figure in liquid medium. The atp7Δ mutant was obtained from the EUROSCARF haploid deletion library in the BY4741 background [28 (link)]. This atp7Δ mutant was complemented with the corresponding wild-type gene expressed from a centromeric plasmid (pRS416), and yeast growth was determined as above.
Complete supplement mixture
Manufactured by Formedium
Sourced in United Kingdom
Complete supplement mixture provides a comprehensive blend of essential nutrients for laboratory applications. This product contains a carefully formulated combination of vitamins, minerals, and other essential compounds to support various experimental requirements.
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Lab products found in correlation
Yeast nitrogen base, by Merck Group (1 mentions)
Glucose, by BD (1 mentions)
Ammonium sulfate, by BD (1 mentions)
Yeast nitrogen base, by BD (1 mentions)
Peptone, by BD (1 mentions)
Yeast extract, by BD (1 mentions)
Ynb without amino acids, by Formedium (1 mentions)
Ethanol, by Merck Group (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Lactate, by Merck Group (1 mentions)
C glabrata atcc 2001, by American Type Culture Collection (1 mentions)
Yeast nitrogen base, by Formedium (1 mentions)
Agar, by Thermo Fisher Scientific (1 mentions)
Uracil drop out, by Qbiogene (1 mentions)
Histidine drop out, by Formedium (1 mentions)
Acetate, by Merck Group (1 mentions)
Candida glabrata atcc 2001, by American Type Culture Collection (1 mentions)
7 protocols using complete supplement mixture
1
Yeast Growth Assay with Edelfosine
2
Yeast Deletion Strain Protocol
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This study used Saccharomyces cerevisiae BY4741 as the wild type background strain, and deletion mutants from the Yeast Deletion Collection30 (link). YPD media; 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose. CSM media; 6.7 g/L yeast nitrogen base (Sigma Y0626), 790 mg/L Complete Supplement Mixture (Formedium DCS001), 20 g/L glucose.
3
Carbon Source Evaluation for Candida glabrata
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The reference strain C. glabrata ATCC 2001 was used in this study (American Type Culture Collection, Manassas, VA, USA). Standard culture media were used, including YPD (Becton, Dickinson and Company, Franklin Lakes, NJ, USA): yeast extract (1%, w/v), peptone (2%, w/v), glucose (2%, w/v), agar (1.5%, w/v), and yeast nitrogen base (YNB) without amino acids (Becton, Dickinson and Company, USA): yeast nitrogen base (0.67%, w/v), ammonium sulfate (0.5%, w/v). Synthetic complete (SC) media were prepared with YNB without amino acids, supplemented with complete supplement mixture (0.2%, w/v) (Formedium, Hunstanton, UK), glucose (2%, w/v), and agar (2%, w/v). In addition, glucose was replaced with alternative carbon sources: acetate (2%, w/v), lactate (2%, v/v), ethanol (2%, v/v), glycerol (2%, v/v), or oleic acid (0.2%, w/v) (Sigma-Aldrich, St. Louis, MO, USA) as the sole carbon source in SC media [2 (link),51 (link)].
4
Yeast Growth in Carbon Sources
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A 10 μL aliquot of the yeast mixture (see above) was added to 10 mL liquid medium. This starting culture was equally divided into four 13 mL round‐bottom tubes (Semadeni Plastics Market, Ostermundigen, Switzerland), and the cultures were kept on a shaking incubator (27°C, 250 rpm; Stuart orbital incubator S150, Staffordshire, UK). At each timepoint, the OD600 of each culture was determined, 0.1 ODU was collected, a 100‐fold dilution was prepared and 20 μL of the suspension was plated on PDA and incubated at 22°C for 2–3 days. Potato Dextrose Broth (PDB; BD Difco™, Becton, Dickinson and Company, Le Pont de Claix, France) as well as Yeast Nitrogen Base (YNB; Formedium™, Norfolk, UK) were used as the basal media. YNB medium was always supplemented with Complete Supplement Mixture (Formedium™, Norfolk, UK) and different carbon sources [glucose monohydrate, maltose monohydrate, melezitose monohydrate and N‐acetyl‐d ‐glucosamine (NAG); all used at 10 g/L and obtained from Carl Roth GmbH, Karlsruhe, Germany].
5
Yeast Strain Engineering for Antifungal Studies
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The S. cerevisiae AD2Δ host was used to create the strains used in this study. The strain was made by deletion of the HIS1 gene from the previously described ADΔ49 (link) strain. ADΔ and AD2Δ lack 7 major ATP-binding cassette transporters and the PDR3 gene. Both strains have the gain-of function pdr1–3 mutation in the PDR1 transcriptional regulator gene to provide constitutive overexpression of ScErg11p6 × His from the PDR5 locus. Yeast strains were grown on YPD medium: 1% (wt/vol) Bacto-yeast extract (BD Difco™ Laboratories Inc, Franklin Lakes, NJ), 2% (wt/vol) Bacto-peptone (BD Difco™) and 2% (wt/vol) glucose. Synthetic defined (SD) medium was used for selection of transformants. It contained 2% (wt/vol) glucose, 0.67% (wt/vol) yeast nitrogen base without amino acids (BD Difco™), 2% (wt/vol) agar (Oxoid Ltd., Hampshire, UK) and either uracil drop-out (Qbiogene, Irvine, CA) or histidine drop-out (Formedium™, Norfolk, UK) complete supplement mixture. Liquid SD media with complete supplement mixture (Formedium™) containing 10 mM MES and 20 mM HEPES buffered with TRIS to pH 6.8 was used for MIC80 determinations.
6
Cultivation of D. hansenii Strain CBS 767
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The D. hansenii strain CBS 767 (PYCC2968; Prista et al., 1997 (link); Navarrete et al., 2009 (link), 2020 (link)) was used in this study. The strain was stored at −80°C in glycerol stocks containing sterile 30% glycerol (Sigma‐Aldrich, Germany).
Cells were grown from the cryostocks at 28°C in Yeast extract Peptone Dextrose (YPD) medium plates with 2% agar. For the pre‐cultures of yeast cells, synthetic complete medium (YNB) was used (6.7 g l‐1 Yeast Nitrogen Base w/o amino acids, from Difco, plus 0.79 g l‐1 complete supplement mixture, from Formedium). Separately sterilized 2% D‐(+)‐glucose monohydrated (VWR Chemicals, VWR International, Darmstadt, Germany) was added to the medium, and the pH was adjusted to 6.0 with NaOH. All the solutions were autoclaved at 121°C for 20 min. Pre‐cultures of 100 ml cell culture were incubated in 500 ml baffled Erlenmeyer shake flasks at 28°C, 150 rpm for at least 24 h.
Cells were grown from the cryostocks at 28°C in Yeast extract Peptone Dextrose (YPD) medium plates with 2% agar. For the pre‐cultures of yeast cells, synthetic complete medium (YNB) was used (6.7 g l‐1 Yeast Nitrogen Base w/o amino acids, from Difco, plus 0.79 g l‐1 complete supplement mixture, from Formedium). Separately sterilized 2% D‐(+)‐glucose monohydrated (VWR Chemicals, VWR International, Darmstadt, Germany) was added to the medium, and the pH was adjusted to 6.0 with NaOH. All the solutions were autoclaved at 121°C for 20 min. Pre‐cultures of 100 ml cell culture were incubated in 500 ml baffled Erlenmeyer shake flasks at 28°C, 150 rpm for at least 24 h.
7
Cultivation of Candida glabrata
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Candida glabrata ATCC 2001 (American Type Culture Collection, USA) was used throughout this study. Standard culture media were used, including YPD (Becton, Dickinson and Company, USA): yeast extract (1%, w/v), peptone (2%, w/v), glucose (2%, w/v), agar (1.5%, w/v) and YNB without amino acids (Becton, Dickinson and Company, USA): yeast nitrogen base (0.67%, w/v), ammonium sulfate (0.5%, w/v). Synthetic complete (SC) media were prepared with YNB without amino acids, supplemented with complete supplement mixture (0.2%, w/v) (Formedium, UK), glucose (2%, w/v) or acetate (2%, w/v) (Sigma-Aldrich, USA) as the sole carbon source.
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