Subcellular Fractionation of Tissue Culture Cells

Fernando Aniento1, Jean Gruenberg2

1 University of Valencia, Valencia, Spain, 2 University of Geneva Sciences II, Geneva, Switzerland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.1C
DOI:  10.1002/0471142735.im0801cs57
Online Posting Date:  November, 2003
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Development of cell fractionation techniques over the last few decades has provided the means to analyze the composition and properties of purified cellular elements. In particular, subcellular fractionation is essential for the development of cell‐free assays that reconstitute complicated cellular processes. These assays have provided new and important tools to understand the molecular mechanisms of complex cellular functions, permitting these functions to be studied in the test tube as a series of biochemical reactions. The protocols in this unit describe fractionation of tissue culture cells to immunoisolate early and late endosomes under conditions where these compartments retain their capacity to support membrane transport in vitro.

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Table of Contents

  • Basic Protocol 1: Internalization of Fluid‐phase Marker
  • Basic Protocol 2: Implantation and Internalization of VSV G Protein
  • Support Protocol 1: Preparation of Vesicular Stomatitis Virus Stocks
  • Basic Protocol 3: Homogenization of Tissue Culture Cells
  • Support Protocol 2: Determination of Horseradish Peroxidase Activity
  • Basic Protocol 4: Flotation‐Gradient Fractionation of Tissue Culture Homogenates
  • Basic Protocol 5: Immunoisolation of Endosomal Fractions
  • Support Protocol 3: Preparation of Tissue Culture Cells for Subcellular Fractionation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Internalization of Fluid‐phase Marker

  • Monolayer cultures of BHK‐21 cells in 10‐cm tissue culture plates, 14 to 18 hr after plating (see protocol 8)
  • PBS ( appendix 2A), ice cold
  • 2 to 10 mg/ml horseradish peroxidase (HRP) in internalization medium (see recipe), 37°C
  • PBS/BSA: PBS containing 5 mg/ml BSA, ice cold and 37°C
  • IM/BSA: Internalization medium (see recipe) containing 2 mg/ml BSA, 37°C
  • Cold plate: wet metal plate lying flat on top of ice in an ice bucket
  • Rocking platform
  • Warm plate: 37°C water bath with a flat metal plate slightly below the water level or 37°C incubator

Basic Protocol 2: Implantation and Internalization of VSV G Protein

  • Monolayer cultures of BHK‐21 cells in 10‐cm tissue culture plates, 14 to 18 hr after plating (see protocol 8)
  • 100 µg/ml wheat germ agglutinin (WGA) from Triticum vulgaris (Sigma) in 7.4 M medium (see recipe), ice cold
  • 20 µg/ml vesicular stomatitis virus (VSV; see protocol 3) in 7.4 M medium (see recipe), ice cold
  • Fusion medium: MEM containing 20 mM succinate, pH 4.9, 37°C
  • 60 mM N‐acetylglucosamine (GlcNAc) in 7.4 M medium (see recipe), ice cold
  • Vacuum aspirator connected to a trap containing disinfectant (e.g., chloramine T)
  • Additional reagents and equipment for fluid‐phase internalization (see protocol 1)
CAUTION: VSV is a rodent pathogen. Consult with the local hazardous materials authority for proper handling and disposal procedures. All plastic, glassware, and solutions can be disinfected with chloramine T.

Support Protocol 1: Preparation of Vesicular Stomatitis Virus Stocks

  • Monolayer cultures of BHK‐21 cells labeled with VSV G protein and/or fluid‐phase marker (see Basic Protocols protocol 11 and protocol 22)
  • PBS ( appendix 2A), 4°C
  • Homogenization buffer (HB; see recipe)
  • Protease inhibitor cocktail (see recipe)
  • 15‐ml plastic conical centrifuge tube
  • Refrigerated centrifuge and rotor appropriate for cell sedimentation
  • 22‐G needle attached to 1‐ml syringe
  • Phase‐contrast microscope
  • Beckman Airfuge or equivalent ultracentrifuge
  • Additional reagents and equipment for assay of horseradish peroxidase activity (see protocol 5)

Basic Protocol 3: Homogenization of Tissue Culture Cells

  • Subcellular fraction containing internalized HRP (see protocol 4)
  • HRP substrate solution (see recipe)
  • HRP standards: 10 to 100 ng HRP/ml in the same buffer used to prepare or dilute sample
  • 10 µM potassium cyanide (KCN; optional)

Support Protocol 2: Determination of Horseradish Peroxidase Activity

  • Postnuclear supernatant (PNS; see protocol 4)
  • Sucrose solutions (see reciperecipes)
  • Homogenization buffer (HB; see recipe)
  • Refractometer
  • SW60 centrifuge tubes
  • Ultracentrifuge and SW60 rotor
  • Peristaltic pump connected to 50‐µl capillary tube

Basic Protocol 4: Flotation‐Gradient Fractionation of Tissue Culture Homogenates

  • Rabbit anti‐mouse immunoglobulin covalently coupled to polyacrylamide or magnetic beads (e.g., Immunobeads; Bio‐Rad)
  • PBS/BSA: PBS ( appendix 2A) containing 2 mg/ml BSA, ice cold
  • Mouse antibody against the cytoplasmic domain of VSV G protein (P5D4; Kreis, )
  • Endosomal fractions (see protocol 6)
  • Microcentrifuge, refrigerated
  • Low‐speed rotating wheel (2 to 20 rpm)
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Literature Cited

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