Dataset Information

Automated image analysis of stained cytospins to quantify Schwann cell purity and proliferation.

ABSTRACT: In response to injury, adult Schwann cells (SCs) re-enter the cell cycle, change their expression profile, and exert repair functions important for wound healing and the re-growth of axons. While this phenotypical instability of SCs is essential for nerve regeneration, it has also been implicated in cancer progression and de-myelinating neuropathies. Thus, SCs became an important research tool to study the molecular mechanisms involved in repair and disease and to identify targets for therapeutic intervention. A high purity of isolated SC cultures used for experimentation must be demonstrated to exclude that novel findings are derived from a contaminating fibroblasts population. In addition, information about the SC proliferation status is an important parameter to be determined in response to different treatments. The evaluation of SC purity and proliferation, however, usually depends on the time consuming, manual assessment of immunofluorescence stainings or comes with the sacrifice of a large amount of SCs for flow cytometry analysis. We here show that rat SC culture derived cytospins stained for SC marker SOX10, proliferation marker EdU, intermediate filament vimentin and DAPI allowed the determination of SC identity and proliferation by requiring only a small number of cells. Furthermore, the CellProfiler software was used to develop an automated image analysis pipeline that quantified SCs and proliferating SCs from the obtained immunofluorescence images. By comparing the results of total cell count, SC purity and SC proliferation rate between manual counting and the CellProfiler output, we demonstrated applicability and reliability of the established pipeline. In conclusion, we here combined the cytospin technique, a multi-colour immunofluorescence staining panel, and an automated image analysis pipeline to enable the quantification of SC purity and SC proliferation from small cell aliquots. This procedure represents a solid read-out to simplify and standardize the quantification of primary SC culture purity and proliferation.

SUBMITTER: Weiss T

PROVIDER: S-EPMC7244157 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Automated image analysis of stained cytospins to quantify Schwann cell purity and proliferation.

Weiss Tamara T Semmler Lorenz L Millesi Flavia F Mann Anda A Haertinger Maximilian M Salzmann Manuel M Radtke Christine C

PloS one 20200522 5

In response to injury, adult Schwann cells (SCs) re-enter the cell cycle, change their expression profile, and exert repair functions important for wound healing and the re-growth of axons. While this phenotypical instability of SCs is essential for nerve regeneration, it has also been implicated in cancer progression and de-myelinating neuropathies. Thus, SCs became an important research tool to study the molecular mechanisms involved in repair and disease and to identify targets for therapeuti ...[more]

PMID: 32442229

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Project description:BackgroundGenome-wide mutant strain collections have increased demand for high throughput cellular phenotyping (HTCP). For example, investigators use HTCP to investigate interactions between gene deletion mutations and additional chemical or genetic perturbations by assessing differences in cell proliferation among the collection of 5000 S. cerevisiae gene deletion strains. Such studies have thus far been predominantly qualitative, using agar cell arrays to subjectively score growth differences. Quantitative systems level analysis of gene interactions would be enabled by more precise HTCP methods, such as kinetic analysis of cell proliferation in liquid culture by optical density. However, requirements for processing liquid cultures make them relatively cumbersome and low throughput compared to agar. To improve HTCP performance and advance capabilities for quantifying interactions, YeastXtract software was developed for automated analysis of cell array images.ResultsYeastXtract software was developed for kinetic growth curve analysis of spotted agar cultures. The accuracy and precision for image analysis of agar culture arrays was comparable to OD measurements of liquid cultures. Using YeastXtract, image intensity vs. biomass of spot cultures was linearly correlated over two orders of magnitude. Thus cell proliferation could be measured over about seven generations, including four to five generations of relatively constant exponential phase growth. Spot area normalization reduced the variation in measurements of total growth efficiency. A growth model, based on the logistic function, increased precision and accuracy of maximum specific rate measurements, compared to empirical methods. The logistic function model was also more robust against data sparseness, meaning that less data was required to obtain accurate, precise, quantitative growth phenotypes.ConclusionMicrobial cultures spotted onto agar media are widely used for genotype-phenotype analysis, however quantitative HTCP methods capable of measuring kinetic growth rates have not been available previously. YeastXtract provides objective, automated, quantitative, image analysis of agar cell culture arrays. Fitting the resulting data to a logistic equation-based growth model yields robust, accurate growth rate information. These methods allow the incorporation of imaging and automated image analysis of cell arrays, grown on solid agar media, into HTCP-driven experimental approaches, such as global, quantitative analysis of gene interaction networks.

Dataset Information

Automated image analysis of stained cytospins to quantify Schwann cell purity and proliferation.

Publications

Automated image analysis of stained cytospins to quantify Schwann cell purity and proliferation.

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