Standard procedure of fission yeast manipulation was followed [80 (link),81 (link)]. Cell growth was carried out in YEA media (3% glucose, 0.5% yeast extract, 75 mg/L adenine) (Sigma-Aldrich, St Louis, MO, USA). Solid media plates contained 2% Bacto-agar (BD Bioscience, Franklin Lakes, NJ, USA). Genetic crosses were performed by mixing haploid cells of opposite mating types on SPA plates and meiotic progenies dissected using automated dissection microscope MSM400 (Singer Instruments, Somerset, UK) as previously described [21 (link),82 (link),83 (link)]. MER strains were obtained from previous screens that examined sensitivity to multiple cytotoxic agents, chemotherapeutic drugs and cation [31 (link),33 (link),34 (link),35 (link)]. These are haploid prototrophic strains and arise from backcrossing commercially purchased single-gene knockout library strains (Bioneer, Daejeon, Republic of Korea) with prototrophic WT strains. Gene disruption was confirmed via PCR using gene-specific primers as described previously [31 (link)]. All strains were stored as a frozen stock in glycerol at −80 °C.
Msm 400
Manufactured by Singer Instruments
Sourced in United Kingdom
The MSM 400 is a benchtop microplate spectrophotometer designed for absorbance measurements. It can accommodate microplates with up to 96 wells and provides a wavelength range of 200 to 1000 nm.
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Lab products found in correlation
14 protocols using msm 400
1
Fission Yeast Genetic Manipulation Protocol
2
Dissection and Genotyping of Sporulated Diploids
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Similarly to above, except that tetrads obtained from sporulation of heterozygous TLC1/tlc1-Δ diploids were manually dissected (MSM 400, Singer Instruments, UK) on YPD plates and left to grow at 30 °C for 2 days. After genotyping of resulting colonies, spot assays were performed as described above. Where indicated, Nicotinamide (NAM, Sigma-Adrich N0636) was added to YPD plates at 5 mM.
3
Monitoring Yeast Cell Growth and Division
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For single-gene copy strains, the log-phase yeast culture was diluted to an OD600 of 0.1 and incubated at 30°C with shaking. Subsequent measurements at OD600 were monitored hourly. In monitoring cell division, single yeast cells were separated via micro-manipulation with the dissection microscope MSM400 (Singer Instruments, Somerset, UK). The number of cells and divisions (per single cell) were then monitored at regular time intervals. For monitoring of cell growth, 0.2 ml of log-phase yeast culture at OD600 of 0.5 was serial diluted in multiples of 5 in the respective growth medium, followed by spotting 2.1 μl of each serial dilution onto the agar plates. Growth of cells on the plates was tracked at 30 or 33°C, over 2–7 days.
4
Replicative Lifespan Analysis in Yeast
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Replicative lifespan was assessed as previously described (69 (link)) using the MSM 400 (Singer Instruments) micromanipulator. Briefly, cells were grown overnight in YPD to early logarithmic phase (A600 = 0.1–0.3), plated, and manually dissected until they did not produce any daughters. Each replicative lifespan analysis was performed independently three times.
5
Phenotypic Changes in E. coli Induced by Honey or Ampicillin
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To examine phenotype changes in E. coli induced by honey or ampicillin at different growth phases, 10 µl samples were removed from the experimental and control wells at each time interval at log-phase and at stationary phase (18 hr incubation) from the 96 well plates. The samples were examined on glass slides at 400 x magnifications under light microscope (Zeiss, Axiolab, Germany). Images were viewed and photographed using the digital camera and built-in software (Singer Instruments MSM 400, Somerset, UK).
6
Rat Erythrocyte Agglutination Assay
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Rat blood collected in tubes containing EDTA was centrifuged at 3,000 × g for 5 min at 4°C. The pellet was washed three times in PBS containing 10 mM EDTA and then with 20 volumes of 75 mM phosphate buffer (pH 7.2) containing 75 mM NaCl. Erythrocytes were re-suspended in PBS as a 10% (v/v) suspension and immediately used in hemagglutination assay. Ten microliters of glycoprotein fractions were mixed with an equal volume of a 2% RBC suspension on glass slides. PBS and phytohemagglutinin were used as a negative and positive control, respectively. Agglutination was observed under light microscope (400x magnification) (Zeiss, Axiolab, Germany) and photographed using the digital camera and built-in software (Singer Instruments MSM 400, Somerset, UK).
7
Standard Yeast Strain Construction
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Standard yeast methods were used to construct yeast strains in this study. All integrated mutations were introduced into the chromosomal loci of each gene of interest and confirmed by DNA sequencing. Double mutants were constructed through genetic crosses and sporulation, yielding multiple independent isolates for genetic analyses. Tetrad dissection was performed on a Singer Instruments MSM 400 and the genotypes of individual spores of each tetrad were confirmed via growth on selective media plates. The strains used are listed in S5 Table , while the plasmids used are listed in S6 Table . Details of plasmid construction are available upon request.
8
Microscopic Analysis of Bacterial Glycoprotein Morphology
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The morphological changes in bacterial cells induced by glycoproteins at different growth phases were examined on glass slides using 10ul samples removed from the experimental and control wells at the time intervals described above. Images were viewed at 400 x magnifications under light microscope (Zeiss, Axiolab, Germany) and photographed using the digital camera and built-in software (Singer Instruments MSM 400, Somerset, UK).
9
Cultivation and Mating of Fission Yeasts
10
Establishing Yeast Virgin Mother Cells
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Yeast cells were grown in YPD at 30 °C overnight, diluted to an OD600 of 0.15, and further grown at 30 °C until OD600 reached 0.5−0.7. The cells were streaked along one side of a YPD agar plate and incubated at 30 °C for 2−3 h. Using a micromanipulation dissection microscope (MSM400, Singer Instruments, United Kingdom), a grid of reference holes was established on the growth surface of the YPD plate. Two or three small healthy yeast cells were transferred to each reference hole. From these cells, 1−3 virgin mother cells per hole were established by separating and discarding mother cells for the first two generations, with the aim of establishing 20−50 virgin mother cells per experiment. Daughter cells were separated and discarded immediately after they budded off. Plates were maintained at permissive temperature (30 °C) and stored at 4 °C overnight throughout the experiment.
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