Live‐Cell Visualization of Calcium Flux in Vibratome‐Cut Thick Sections of Viable Tumor Tissue

James Koh1, Joyce A. Hogue2, Julie A. Sosa3

1 Duke Cancer Institute, Duke University Medical Center, Durham, North Carolina, 2 Department of Surgery, Duke University Medical Center, Durham, North Carolina, 3 Duke Clinical Research Institute, Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.34
DOI:  10.1002/cpcb.37
Online Posting Date:  December, 2017
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Abstract

This unit outlines a live‐cell imaging approach developed for visualization of intracellular calcium flux in human parathyroid tumors following stimulation of the calcium‐sensing receptor (CASR), a class C G protein–coupled receptor (GPCR). The primary assay readout, intracellular calcium release induced by activation of the inositol triphosphate receptor, is potentially generalizable to multiple other GPCR signaling events that utilize this common downstream signal transduction pathway. Advantages of the approach include: (1) preservation of native tissue context and positional information, allowing direct visualization of intratumoral functional heterogeneity; (2) quantitative documentation of reactivity to a physiological stimulus in an experimentally tractable ex vivo system; and (3) generation of a dynamic, functional classifier of tumor biochemical behavior to augment static marker assessment. The technical steps are performed in three sequential phases: (1) viable tissue sectioning; (2) fluorophore loading and tissue immobilization; and (3) live‐cell confocal microscopy. This versatile method provides a straightforward platform for functional characterization of human tumors. © 2017 by John Wiley & Sons, Inc.

Keywords: calcium flux; live‐cell imaging; viable tissue sectioning; signal transduction methods

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Tissue Embedding and Sectioning
  • Basic Protocol 2: Tissue Slice Culture Incubation, Fluo‐4 Loading, and Immobilization
  • Basic Protocol 3: Live‐Cell Confocal Imaging
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Tissue Embedding and Sectioning

  Materials
  • Tumor tissue of interest
  • Dulbecco's Modified Eagle Medium (DMEM; e.g., Thermo Fisher Scientific, cat. no. 11885)
  • Antibiotic/Antimycotic solution (100×; e.g., Thermo Fisher Scientific, cat. no. 15240062)
  • Low‐melting‐temperature agarose (e.g., BioExpress, cat. no. E‐3126‐25)
  • Phosphate‐buffered saline, sterile (PBS; e.g., Thermo Fisher Scientific, cat. no., 14190)
  • Loctite tissue adhesive (e.g., Ted Pella, cat. no. 10035)
  • Sterile conical tube, appropriately sized for tissue of interest
  • Laminar flow hood
  • 35‐mm tissue culture dishes (e.g., VWR, cat. no. 25382‐348)
  • Scalpel, for trimming tissue
  • Forceps
  • Lint‐free absorbent tissue
  • Vibratome (e.g., Leica VT1000S or equivalent)
  • Vibratome blades (e.g., Ted Pella, cat. no. 121‐19)
  • Autoclavable histology brushes, sterile (e.g., Gordon Brush, cat. no. 250NAD)
NOTE: All procedures involving procurement of human tissue must first be reviewed and approved by an Institutional Review Board, and tissue may only be collected after obtaining the patient's written informed consent.

Basic Protocol 2: Tissue Slice Culture Incubation, Fluo‐4 Loading, and Immobilization

  Materials
  • Culture medium optimized for tissue source
  • Tissue slices (see protocol 1)
  • Fluo‐4‐AM NW Assay Kit (e.g., Thermo Fisher Scientific, cat. no. F14201)
  • Calcium‐free Hank's balanced salt solution (HBSS; e.g., Thermo Fisher Scientific, cat. no. 14175)
  • Hoechst 33342 cell permeant nuclear dye (e.g., Sigma‐Aldrich, cat. no. 14533)
  • Low‐melting‐temperature agarose (e.g., BioExpress, cat. no. E‐3126‐25)
  • Multiwell tissue culture plate
  • Transwell culture inserts (3‐µm pore size; e.g., VWR, cat. no. 10769‐194)
  • Autoclavable histology brushes, sterile (e.g., Gordon Brush, cat. no. 250NAD)
  • Humidified, CO 2, water‐jacketed tissue culture incubator
  • 35‐mm tissue culture dishes (e.g., VWR, cat. no. 25382‐348)
  • 250‐µm pore size Nitex nylon mesh (e.g., Genesee Scientific, cat. no. 57‐107)
  • 1‐ml syringe
  • Forceps

Basic Protocol 3: Live‐Cell Confocal Imaging

  Materials
  • Fluo‐4‐AM loaded tissue (see protocol 2)
  • Calcium agonist: CaCl 2 in HBSS
  • Ionomycin (e.g., Thermo Fisher Scientific, cat. no. 124222)
  • 1 mg/ml propidium iodide (PI; e.g., Thermo Fisher Scientific, P3566)
  • Upright confocal microscope (e.g., Zeiss LSM780 or equivalent), fitted with a 20× water immersion objective lens (e.g., W Plan‐Apochromat, 20×/1.0, WD 1.8 mm)
  • Image analysis software (e.g., Fiji or CellProfiler)
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Figures

Videos

Literature Cited

Literature Cited
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  Koh, J., Hogue, J. A., Wang, Y., DiSalvo, M., Allbritton, N. L., Shi, Y., … Sosa, J. A. (2016). Single‐cell functional analysis of parathyroid adenomas reveals distinct classes of calcium sensing behaviour in primary hyperparathyroidism. Journal of Cellular and Molecular Medicine, 20, 351–359. doi: 10.1111/jcmm.12732.
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  Shi, Y., Hogue, J., Dixit, D., Koh, J., & Olson, J. A., Jr. (2014). Functional and genetic studies of isolated cells from parathyroid tumors reveal the complex pathogenesis of parathyroid neoplasia. Proceedings of the National Academy of Sciences of the United States of America, 111, 3092–3097. doi: 10.1073/pnas.1319742111.
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