Isolation of Endothelial Cells from Mouse Lung

Gaoyuan Cao1, Valsamma Abraham1, Horace M. DeLisser1

1 Pulmonary, Allergy and Critical Care Division, Department of Medicine, Perelman School of School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 24.2
DOI:  10.1002/0471140856.tx2402s61
Online Posting Date:  August, 2014
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Abstract

The isolation of endothelial cells (ECs) from knockout and transgenic mouse lines provides the opportunity to study the endothelial‐specific activities of a targeted molecule. As a means of pursuing these types of investigations, the protocols described in this unit provide a reliable method for isolating lung microvascular ECs from mouse neonatal pups that can be serially passaged. These protocols are useful in settings where mouse age is irrelevant and a pure population of pulmonary vascular ECs, uncontaminated by other cells, is needed. When a specific source of ECs is not required, these procedures also represent a reliable means of obtaining murine ECs in general. Curr. Protoc. Toxicol. 61:24.2.1‐24.2.9. © 2014 by John Wiley & Sons, Inc.

Keywords: endothelial cells; microvascular cells; lung cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Digestion of the Lung to a Mixed Population of Cells
  • Basic Protocol 2: Generation of a Pure Population of Endothelial Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Digestion of the Lung to a Mixed Population of Cells

  Materials
  • Four to six C57BL/6 neonatal mouse pups (5 to 14 days old)
  • 1000 USP units/ml heparin (Sigma) in phosphate‐buffered saline (PBS; appendix 2A)
  • Ketamine (Butler)
  • Xylazine (AnaSed, Lloyd)
  • 70% ethanol
  • Serum‐free DMEM: Dulbecco's Modified Eagle's medium (DMEM) containing 1× penicillin/streptomycin and 2 mM L‐glutamine
  • DMEM digestion solution: Dulbecco's Modified Eagle's medium (DMEM) containing 1× penicillin/streptomycin, 2 mM L‐glutamine, 20 µg/ml DNase I (Sigma), and 1.0 mg/ml collagenase A (for 7‐ to 10‐day‐old pups) or 1.5 mg/ml collagenase A for 11‐ to 14‐day‐old pups; purchase collagenase A from Roche)
  • Complete DMEM: Dulbecco's Modified Eagle's medium (DMEM) containing 10% FBS, 1× penicillin/streptomycin, and 2 mM L‐glutamine
  • Dissection board
  • Dissection instruments
  • Animal hair clippers
  • 24‐G angiocath
  • 3–0 sutures
  • 70‐µm cell strainer (BD Falcon)
  • Hemacytometer
  • 1% gelatin–coated 75‐cm2 (T‐75) tissue culture flasks (see recipe)
  • Additional reagents and equipment for injection of mice (Donovan and Brown, ), anesthesia of mice (Donovan and Brown, ), and cell culture techniques including counting cells ( appendix 3B)

Basic Protocol 2: Generation of a Pure Population of Endothelial Cells

  Materials
  • 0.05% trypsin/EDTA (Life Technologies, cat. no. 25300‐054)
  • Cell sorting buffer: PBS ( appendix 2A) containing 2% (w/v) BSA
  • Rat anti‐mouse antibodies:
    • Anti‐ICAM‐2 antibody, clone 3C4, unconjugated and fluorescein conjugated (Southern Biotech)
    • Anti‐PECAM‐1 antibody, clone 390, unconjugated and Alexa Fluor 647 conjugated (Southern Biotech)
    • Anti‐VE‐cadherin, unconjugated and phycoerythrin conjugated (BD Biosciences)
  • 7‐amino‐actinomycin D (7‐AAD; BD Biosciences)
  • M199 sorting medium: M199 medium containing 50% (v/v) FBS, 50 µg/ml endothelial growth factor (BD Bioscience), 100 µg/ml heparin, and 1 mM glutamine
  • Complete M199 medium: M199 medium containing 15% (v/v) FBS, 50 µg/ml endothelial growth factor (BD Bioscience), 100 µg/ml heparin, and 1 mM glutamine
  • H5V murine endothelial cell line (Garlanda et al., ; alternative murine endothelial cell lines are available through the ATCC, including the C166, 2H11 and SVEC‐410 lines), cultured in complete M199 medium
  • 3T3 fibroblast line (ATCC #CRL‐1658), cultured in complete M199 medium
  • Enzyme‐free, cell dissociation buffer (Life Technologies)
  • FACS staining buffer: PBS ( appendix 2A) with 2% (v/v) heat inactivated serum and 0.09% sodium azide
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Acetone (Sigma)
  • Methanol (Sigma)
  • Blocking buffer: PBS with 5% normal goat serum and 1% bovine serum albumin (BSA)
  • Rat IgG2a, Alexa Fluor 647 conjugated (used as isotype control for FACS; Southern Biotechnology)
  • Goat anti–rat IgG antibody, Alexa Fluor 594 conjugated (Invitrogen)
  • ProLong Gold antifade reagent with DAPI (Invitrogen)
  • Matrigel (BD Biosciences)
  • 4% paraformaldehyde (Polysciences)
  • Centrifuge
  • FACSVantage SE with DiVa software (BD Bioscience)
  • 1% gelatin–coated 25‐ (T‐25) and 75‐cm2 (T‐75) tissue culture flasks (see recipe)
  • 4‐Chamber CultureSlide (BD Falcon)
  • Nikon Eclipse TE 2000 phase‐contrast microscope
  • Leica TCS SP5 II scanning laser confocal microscope
  • Coverslips (Fisher Scientific, premium coverslips, cat. no. 12‐548‐5M)
  • 96‐well tissue culture plates (BD Falcon)
  • Digital image‐capture system
  • Image‐Pro Plus image analysis software (Media Cybernetics)
  • Additional reagents and equipment for cell culture techniques including counting cells ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
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