Sporulation was set up as described above. After 7 days of sporulation (BY background), 106 cells were pelleted and resuspended in 500 μl of water containing 0.5 mg/ml Zymolyase-100T (Zymo research) and 1% β-mercaptoethanol and incubated overnight at 30°C with gentle rocking to lyse unsporulated diploid cells. A total of 200 μl of 1.5% NP-40 was added to the solution and vortexed to disrupt tetrads (as observed by microscopy). Spores were centrifuged, resuspended in water and plated on YPD plates in serial dilutions to count the number of viable spores (19 (link),20 (link)).
Zymolyase 100t
Manufactured by Zymo Research
Sourced in United States
Zymolyase-100T is a purified enzyme preparation from Arthrobacter luteus. It is primarily used for the lysis of yeast cell walls, enabling the efficient extraction of cellular components such as proteins, RNA, and DNA.
Automatically generated - may contain errors
4 protocols using zymolyase 100t
1
Sporulation and Spore Viability Assay
2
Yeast Cell Membrane Integrity Analysis
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The membrane integrity of yeast was analyzed using a Zymolyase assay. The cultures were supplemented with 1 mg/mL andrographolide and DMSO serving as control and incubated for 16 h at 30 °C with shaking. The stationary phase cultures were then harvested by centrifugation at 6000× g for 5 min at 25 °C, washed once with sterile distilled water, and resuspended in 50 mM potassium phosphate (pH 7.5) and 10 mM 2-mercaptoethanol. Zymolyase-100T (0.4 units) (Zymo Research, Irvine, CA, USA) was added to cell suspensions and the absorbance at 600 nm was measured using a Multiskan SkyHigh (Thermo Fisher Scientific) every 5 min for 1 h [26 (link),27 (link)].
3
Cell Wall Degradation Kinetics
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Strains were grown with their respective controls as described for the expression microarray experiments. Wild-type and ∆prz1 strains were grown in liquid YES medium, while genetic backgrounds containing nmt1-driven prz1+ or empty vector were grown in liquid EMM minus thiamine for 18–24 hr. The cells were washed twice with 0.9% saline solution and resuspended in TE buffer at ∼1.2 × 107 cells/ml. Three milliliters of cell suspension was transferred to test tubes in the presence and absence of 25 U/ml Zymolyase 100T (Zymo Research) and shaken at 37°. OD600 readings were taken every 15 min to assess the degree of cell-wall degradation. The significance of the different treatments was assessed at the 2-hr time point with an ANOVA followed by a two-tailed t-test.
4
Visualizing Phagosome-Lysosome Fusion in Candida-Infected Macrophages
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RAW264.7 cells were co-cultured with C. albicans cells, CFG185 (PUT2/PUT2-HA), for 90 min on glass coverslips at a MOI of 5:1 (C:M). Cells were fixed in 3.7% formaldehyde-PBS for 15 min, and permeabilized in 0.25% Tween-20 for 15 min, both incubations were at room temperature. The fixed and permeabilized cells were incubated in zymolyase buffer (2U zymolyase 100T (Zymo Research, Irvine, CA, USA), 10 mM DTT in PBS) for 1 h at 30 °C. After washing, cells were incubated at room temperature in 0.25% Tween-20 for 10 min and blocked in 5% FBS for 30 min. Cells were incubated overnight at 4 °C with rat anti-HA (Roche, Germany, #1867423) and rabbit anti-Lamp1 (Abcam, UK, #ab24170) primary antibodies diluted 1:500 in 0.25% Tween-20. Cells were washed with PBS and incubated 2 h with Alexa flour 488 goat anti-rabbit (Invitrogen, Eugene, OR, USA #A11034) and Alexa flour 555 goat anti-rat (Invitrogen, Eugene, OR, USA #A11034) secondary antibodies diluted 1:500 in 0.25% Tween-20. Images were captured on a Zeiss 510 Meta confocal microscope, 63x/1.4 oil. Orthogonal views were constructed in FIJI imaging software.
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