High‐Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification

Torsten Kroll1, David Schmidt1, Georg Schwanitz2, Mubashir Ahmad3, Jana Hamann2, Corinne Schlosser2, Yu‐Chieh Lin2, Konrad J. Böhm2, Jan Tuckermann3, Aspasia Ploubidou2

1 These authors contributed equally to this work, 2 Leibniz Institute on Aging—Fritz Lipmann Institute, Jena, 3 Institute for Comparative Molecular Endocrinology, University of Ulm, Ulm
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.43
DOI:  10.1002/cpcy.7
Online Posting Date:  July, 2016
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High‐content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high‐throughput image acquisition, HCA applied to the screening of chemicals or RNAi‐reagents is termed high‐content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step‐by‐step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy‐based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc.

Keywords: focal adhesions; high‐content analysis; high‐content screening; microscopy automation; quantitative microscopy; subcellular organelles

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Table of Contents

  • Introduction
  • Basic Protocol 1: Automated Image Acquisition and Quantitative Analysis of Focal Adhesions
  • Alternate Protocol 1: Alternative Whole Cell/Cell Outline Labeling
  • Alternate Protocol 2: Adaptation of the Image‐Analysis Protocol to Other HCA Applications
  • Support Protocol 1: Data Meta‐Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Automated Image Acquisition and Quantitative Analysis of Focal Adhesions

  • 70% (v/v) ethanol
  • 1× phosphate‐buffered saline (PBS; see recipe for 10× stock solution), sterile
  • African green monkey kidney fibroblast (COS‐7) cells in 10‐cm Petri dishes
  • Trypsin‐EDTA solution (e.g., Biowest, cat. no. L0930‐100)
  • Culture medium: DMEM supplemented with 2 mM l‐glutamine (add from 200 mM stock) and 10% (v/v) FBS
  • Sodium borohydride (NaBH 4; observe precautions described in MSDS)
  • Fixative (see recipe)
  • Blocking buffer: 1% (w/v) BSA and 2% (v/v) FBS in 1× PBS
  • Primary antibodies:
    • Rabbit anti‐α‐tubulin antibody (Abcam, cat. no. ab53866; 1:150 dilution in blocking buffer)
    • Mouse anti‐paxillin antibody (Transduction Lab, cat. no. P13520; 1:1000 dilution in blocking buffer)
  • Secondary antibodies:
    • Goat anti‐rabbit AlexaFluor647‐conjugated IgG antibody (Life Technologies, cat. no. A21244; 1:400 dilution in blocking buffer)
    • Goat anti‐mouse AlexaFluor594‐conjugated IgG antibody (Life Technologies, cat. no. A11005; 1:800 dilution in blocking buffer)
  • 1 μg/ml (w/v) 4′, 6‐diamidino‐2‐phenylindole (DAPI) in distilled, deionized H 2O (observe precautions described in MSDS; store at 4ºC protected from light)
  • Sodium azide (NaN 3; observe precautions described in MSDS)
  • Antifade mounting medium (see recipe)
  • Liquid/cell dispenser unit compatible with microplates, with dispenser cassettes for 1 to 50 µl and 10 µl to 10 ml dispensing volumes (e.g., MicroFlo Select, BioTek Instruments GmbH)
  • Liquid handling workstation (e.g., Freedom Evo, TECAN)
  • Tissue culture‐treated Petri dishes or flasks
  • Epifluorescence microscope with appropriate filters and a CCD camera, either inverted, with long‐distance objectives (for specimens prepared in multiwell plates or on slide‐mounted coverslips) or upright (for specimens prepared on slide‐mounted coverslips)
  • Micro‐plate handler (e.g., Twister II, Perkin‐Elmer)
  • Automated cell counter (e.g., CASY, Schärfe System GmbH) or Neubauer cell counting chamber
  • Reagent troughs (e.g., VWR, cat. no. 613‐0466)
  • Multichannel (8‐ or 12‐channel) dispensing pipettor, 10‐ to 200‐µl step size
  • Tissue culture–treated 96‐ or 384‐well black‐walled assay plates with flat cell attachment surface (“flat bottom” plates; e.g., Corning Falcon, cat. no. 353219)
  • Adhesive foil microtiter plate sealers (e.g., ViewSeal; Greiner, cat. no. 676070)
  • Automated epifluorescence microscope (e.g., GE IN Cell Analyzer 2200, Thermo Cellomics ArrayScan VTI, BD Pathway 435) with a set of fluorescence filters covering the visible and near‐infrared range of the light spectrum, as well as multiple objectives (e.g., 10×, 20×, 40×)
  • Microscope‐associated image‐acquisition software
  • Image analysis software (e.g., CellProfiler; Carpenter et al., ; Kamentsky et al., )
  • Image‐acquisition and data‐analysis workstations (for additional information, see Internet Resources; in this protocol, image analysis was performed with CellProfiler and data meta‐analysis with Microsoft Excel, R, STRING, DAVID, and Cytoscape
  • Glass coverslips (thickness #1; 12‐mm diameter; e.g., VWR Menzel‐Gläser, cat. no. 631‐0713)
  • Glass microscope slides (e.g., Carl Roth GmbH & Co. KG, cat. no. H868)
  • Additional reagents and equipment for counting cells ( appendix 3A; Phelan and Lawler, )
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