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1.
Fig. 7.3

Fig. 7.3. From: High-Throughput Immunofluorescence Microscopy Using Yeast Spheroplast Cell-Based Microarrays.

Examples of microtubule and spindle morphology in different yeast cell cycle stages from (a) to (d) show G1 phase cells, S/G2 phase cells, anaphase cells, and completed cytokinesis, respectively. Nuclei and microtubules are depicted.

Wei Niu, et al. Methods Mol Biol. ;706:83-95.
2.
Fig. 7.4

Fig. 7.4. From: High-Throughput Immunofluorescence Microscopy Using Yeast Spheroplast Cell-Based Microarrays.

Example images for TetO7 promoter strains exhibiting defective microtubule and spindle structure (a, b). Down-regulation of SPC97 and SPC98 resulted in short spindles and elongated cytoplasmic microtubules (c). Downregulation of TID3 caused defective spindle elongation and uneven nuclear division.

Wei Niu, et al. Methods Mol Biol. ;706:83-95.
3.
Fig. 7.2

Fig. 7.2. From: High-Throughput Immunofluorescence Microscopy Using Yeast Spheroplast Cell-Based Microarrays.

A microarray printing robot suitable for printing yeast spheroplast microarrays, shown alongside an example microarray (a). A Stanford-style Newport DNA microarray print robot with 12 pins directly delivers yeast spheroplasts from 96-well plate onto slides (a) (). A yeast spheroplast array carrying ∼700 yeast TetO7-promoter strains (spot diameter, 200 μm; spot-to-spot distance, 410 μm) is shown in (b).

Wei Niu, et al. Methods Mol Biol. ;706:83-95.
4.
Fig. 7.6

Fig. 7.6. From: High-Throughput Immunofluorescence Microscopy Using Yeast Spheroplast Cell-Based Microarrays.

Spheroplast microarrays can be stored at least a month at −80°C without substantial effects on cellular morphology or subcellular structure, as shown here for microtubule structure. Compared to fresh chips (a), chips stored at −80°C still maintain intact microtubule structures after one month (b). Nuclei and microtubules are depicted.

Wei Niu, et al. Methods Mol Biol. ;706:83-95.
5.
Fig. 7.1

Fig. 7.1. From: High-Throughput Immunofluorescence Microscopy Using Yeast Spheroplast Cell-Based Microarrays.

Work flow of the yeast spheroplast microarray protocol. Yeast cells are cultured, fixed, and spheroplasted in 96-well plates, e.g., using an automated liquid handling robot. Subsequently, they are robotically printed onto poly-l-lysine microscope slides using a slotted steel pin-based DNA microarray robot. The resulting slides each contain ∼5,000 spots. Each slide can be stored at −80°C or can be probed with a specific antibody immediately after printing. Images are acquired through automated microscopy. The different steps of the protocol are indicated in boxes by arrows. The approximate time required for each step is indicated near the box. Steps at which the protocol can be paused are indicated in the diagram by pairs of short lines across the arrows. Times indicated for each step are approximate and will depend upon the precise equipment used for the experiment.

Wei Niu, et al. Methods Mol Biol. ;706:83-95.
6.
Fig. 7.5

Fig. 7.5. From: High-Throughput Immunofluorescence Microscopy Using Yeast Spheroplast Cell-Based Microarrays.

Examples of morphology changes of yeast cells treated with zymolyase to differing extents. Cells should be a dark, translucent grayafter an appropriate degree of digestion (d). (). Bright cellsare insufficiently digested (ac) (). “Ghost cells” (pale gray with little any internal structure) have been over-digested (e, f) (). Spheroplasting protocols for cells typically employ a relatively high concentration of zymolyase and short digestion times (less than 30 min). However, given that yeast spheroplast microarrays are designed for large-scale experiments usually analyzing hundreds or thousands strains at a time, 30 min digestion times are impractical and unforgiving for small errors in timing. We therefore employed lower concentrations of zymolyase and longer digestion times in order to get an appropriate degree of digestion. The optimal concentration for the TetO7 strain collection in our example assay was 0.025 mg/ml; the digestion time was 2 h (d).

Wei Niu, et al. Methods Mol Biol. ;706:83-95.

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