The potential of the most potent extract with antioxidant activity in the protection of yeast cells against induced oxidant stress was evaluated (Koziol et al. 2005 (link)). The wild-type SP-4 (MATα leu1 arg4) of Saccharomyces cerevisiae and its CuZnSOD disruptant sod1::natMX were obtained from Prof. S. Bednarska from University of Rzeszow. Dilutions (1 × 104, 1 × 103, 1 × 102, 10 cells/mL) of yeast exponential phase cultures in a volume of 10 µL were inoculated on solid YPD medium (Sigma, Y-1375), with or without an addition of 0.8 M NaCl and antioxidant extract. Stock solutions of extract and ascorbic acid (as a positive control) were added to sterile media at a final concentration of 1 mg/mL. The media without addition of extract and with an addition of the extract from uninoculated fermentation medium were considered as blank and negative control, respectively. Media were cooled to just above the solidification point before addition of antioxidants. Plates were incubated at 28 °C and examined after 72 h.
Y1375
Manufactured by Merck Group
Sourced in United States
The Y1375 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose measurement device for scientific applications. The core function of the Y1375 is to provide accurate and reliable measurements across various parameters required in laboratory settings.
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6 protocols using y1375
1
Antioxidant protection of yeast cells
2
Air-Drying Budding Yeast for Spaceflight
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The method to air-dry budding yeast cells for the BioSentinel mission has been described previously (Santa Maria et al., 2020 (link)). Briefly, yeast from frozen glycerol stocks were grown on yeast extract-peptone-dextrose (YPD) agar (Y1500; Sigma-Aldrich) for 2–3 days at 30°C. Samples from freshly grown patches were then inoculated into 5 mL of liquid YPD (Y1375; Sigma-Aldrich) and grown on a rotating mixer at ambient temperature (∼23°C) for 7 days. Liquid cultures were pelleted and washed with sterile water, and the cell density was determined by hemacytometer counting. Cell suspensions were prepared to a final density of 1 × 107 cells in 1 mL of 10% trehalose (T0167; Sigma-Aldrich). Ten microliter aliquots of the 1 × 107 cell suspension (containing ∼105 cells) were gently dispensed into the bottom edge of the wells of 96-well Stripwell™ microplates (9102; Costar®) or onto the sidewall of each microfluidic card well (Padgen et al., 2021 (link)).
Plates were sealed with Breathe-Easy® gas permeable sealing membrane (Z380059; Sigma-Aldrich), and the cells were air dried in a 23°C incubator, 20–30% relative humidity, for 7 days. Yeast cells in fluidic cards were air dried inside sterile boxes at the same conditions before card sealing using a pneumatic press (Padgen et al., 2021 (link)).
Plates were sealed with Breathe-Easy® gas permeable sealing membrane (Z380059; Sigma-Aldrich), and the cells were air dried in a 23°C incubator, 20–30% relative humidity, for 7 days. Yeast cells in fluidic cards were air dried inside sterile boxes at the same conditions before card sealing using a pneumatic press (Padgen et al., 2021 (link)).
3
Fungal Load Quantification in Fly Larvae
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Following infection, larvae were washed in 100% ethanol, rinsed in sterile water and transferred to normal fly food. Larvae were homogenized (at indicated time points) in 200 µL YPD media (Y1375, Sigma-Aldrich, USA). Serial dilutions were made and 50 µL, plated on YPD plates (Y1500, Sigma-Aldrich, USA). Plates were incubated at 30o and colonies were measured accordingly.
4
Viability Assessment of E. coli and Yeast Cells
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E. coli are routinely cultured in Luria-Bertani (LB) medium (Sigma Aldrich, L3522), which consists of 10 g L−1 tryptone, 5 g L−1 yeast extract, and 5 g L−1 NaCl. The culture is shaken at 230 rpm at 37 °C until OD reaches 0.7. Yeast cells are routinely cultured in yeast extract–peptone–dextrose (YPD) medium (Sigma Aldrich, Y1375, 10 g L−1 yeast extract, 20 g L−1 bacteriological peptone, and 20 g L−1 glucose). The resulting E. coli and yeast cells are transferred into DI water by centrifugation and redispersion processes (three times) and mixed with synthetic particles. Immediately, a total of 25 μL of the mixed suspension is injected into the chamber made by ITO glass and AC electric field is applied to perform the assembly. The viability of cells after assembly experiment are checked by extracting cells and stained with Trypan blue, which diffuse in dead cells but not live ones. The number of viable and dead cells are counted based on optical microscope images. Cells are subject to electric field for the course of 3, and 30 min for the analysis.
5
Preparation of Heat-Killed Candida albicans
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C. albicans strain SC5314 was provided by Dr. Changbin Chen (Institute Pasteur of Shanghai, CAS, Shanghai, China). Single colonies of C. albicans strain SC5314 from yeast-peptone-dextrose (YPD: Y1375, Sigma) agar plates were inoculated into YPD medium by culture overnight at 30 °C. Yeast cells were cultured at 37 °C for 3 h in YPD medium plus 10% FBS to obtain hyphae. Yeast or Hyphae cells were washed three times and resuspended in PBS buffer, then were incubated at 65 °C for 1 h to kill cells, then obtained HKCA-Y or HKCA-H. The death of Heat-killed yeast or Hyphae cells was verified by plating cells on YPD agar plates.
6
Desiccation Resistance Assay for Yeast Strains
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Wild type and rad51Δ diploid strains from −80°C glycerol stocks were plated on yeast extract - peptone - dextrose (YPD) agar (Sigma-Aldrich; Y1500) and incubated at 30°C for 2–3 days. Samples from freshly grown patches were then used to inoculate 5-mL YPD liquid cultures (Sigma-Aldrich; Y1375), which were grown on a rotating mixer at ambient room temperature (∼23°C) for 7 days. Liquid cultures were pelleted and washed with 1 mL sterile deionized water and then resuspended in 1 mL sterile deionized water. Cell density was determined by hemocytometer counting, and suspensions were diluted to a final density of 1 × 107 cells/mL in 10% (w/v) trehalose (Sigma-Aldrich; T0167). Ten microliters of trehalose suspensions (containing ∼105 cells) were gently dispensed into the bottom edge of the wells of 96-well Stripwell™ microplates (Costar®; 9102). Undesiccated controls were plated on YPD agar (Sigma-Aldrich; Y1500) for colony counting.
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