Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient Centrifugation

John M. Graham1

1 Liverpool John Moores University, Liverpool
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.6
DOI:  10.1002/0471143030.cb0306s07
Online Posting Date:  May, 2001
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Abstract

This unit covers the use of Percoll, Nycodenz, and iodixanol for purification of lysosomes from a light mitochondrial pellet or postnuclear supernatant. The first protocol describes isolation of lysosomes from rat liver using a Percoll gradient; it includes some comments on using brain and kidney tissue as source material. Alternatively, this protocol can be modified to enhance the separation of lysosomes by organelle density perturbation. Another alternative uses a discontinuous gradient of Nycodenz to purify lysosomes from a rat liver light mitochondrial pellet. A continuous iodixanol gradient, which can be used to purify other organelles in the light mitochondrial fraction, is yet another alternative for purifying lysosomes. Lysosomes can also be isolated from some of the more commonly used cultured cells. The unit also includes assays for common lysosome marker enzymes.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Lysosomes from Rat Liver Using a Self‐Generated Percoll Gradient
  • Alternate Protocol 1: Isolation of Lysosomes from Rat Liver Using Density Perturbation and a Self‐Generated Percoll Gradient
  • Alternate Protocol 2: Isolation of Lysosomes from a Rat Liver Light Mitochondrial Fraction in a Discontinuous Gradient of Nycodenz
  • Alternate Protocol 3: Fractionation of a Rat Liver Light Mitochondrial Fraction in a Continuous Iodixanol Gradient
  • Basic Protocol 2: Isolation of Lysosomes from Human HL‐60 Cultured Cells Using a Self‐Generated Percoll Gradient
  • Support Protocol 1: Assay for Acid Phosphatase
  • Support Protocol 2: Assay for β‐N‐Acetylglucosaminidase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Lysosomes from Rat Liver Using a Self‐Generated Percoll Gradient

  Materials
  • 150‐ to 200‐g Sprague‐Dawley rats
  • Homogenization medium (HM; see recipe)
  • Percoll stock solution (see recipe)
  • Dissecting equipment
  • ∼40‐ and ∼5 ml Dounce homogenizers with loose‐fitting Wheaton type B pestles (e.g., Kontes)
  • Low‐speed centrifuge with swinging‐bucket rotor to take 50‐ml tubes
  • Sorvall RC series centrifuge and SE‐12 and SS‐34 rotors or equivalents
  • Gradient unloader: thin metal cannula connected to a peristaltic pump
  • Ultracentrifuge with fixed‐angle rotor (e.g., Beckman 50 Ti or 70.1 Ti or Sorvall T‐865.1)
NOTE: Protease inhibitors (unit 3.4) can be added to any or all of the solutions.

Alternate Protocol 1: Isolation of Lysosomes from Rat Liver Using Density Perturbation and a Self‐Generated Percoll Gradient

  • 100 mg/ml dextran solution (see recipe)
  • Homogenization medium (HM; see recipe) without EDTA
  • 100 mM CaCl 2 (see recipe)
  • Percoll stock solution (see recipe) without EDTA
  • 2‐ml syringe and 23‐G needle
  • Beckman 70.1 Ti rotor

Alternate Protocol 2: Isolation of Lysosomes from a Rat Liver Light Mitochondrial Fraction in a Discontinuous Gradient of Nycodenz

  • 45% Nycodenz stock solution (see recipe)
  • Ultracentrifuge with swinging‐bucket rotor to take ∼38‐ml tubes (e.g., Beckman SW28, Sorvall AH629, or equivalent)
  • Gradient unloader: 1‐ to 2‐ml syringe attached to metal cannula (i.d. 1 mm)

Alternate Protocol 3: Fractionation of a Rat Liver Light Mitochondrial Fraction in a Continuous Iodixanol Gradient

  • OptiPrep diluent (see recipe)
  • OptiPrep (Accurate Chemical)
  • Dense gradient unloading liquid (e.g., Maxidens; Lipotek, U.K.)
  • Two‐chamber gradient maker or Gradient Master
  • Gradient unloader
  • Ultracentrifuge with Beckman SW28.1 or equivalent swinging bucket rotor

Basic Protocol 2: Isolation of Lysosomes from Human HL‐60 Cultured Cells Using a Self‐Generated Percoll Gradient

  Materials
  • HL‐60 cells (ATCC#CCL‐240) in recipehomogenization medium
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Homogenization medium (HM; see recipe)
  • 10% (w/v) bovine serum albumin (BSA)
  • Percoll stock solution
  • 10% (w/v) Nonidet‐40 (NP‐40) in water
  • Ball‐bearing cell homogenizer (see Balch and Rothman, , for more details)
  • Low‐speed centrifuge with swinging‐bucket rotor to take 10‐ml tubes
  • Sorvall high‐speed centrifuge with SE‐12 and SS‐34 or equivalent fixed‐angle rotors
  • Beckman ultracentrifuge with 50Ti, 70Ti, T865 (Sorvall), or equivalent fixed‐angle rotor
  • Gradient unloader: thin metal cannula connected to a peristaltic pump
NOTE: An ultracentrifuge rotor is required to pellet Percoll (step ), but gradient formation (step ) can be carried out in a high‐speed centrifuge or ultracentrifuge.NOTE: Protease inhibitors (unit 3.4) can be added to any or all of the solutions.

Support Protocol 1: Assay for Acid Phosphatase

  Materials
  • Assay buffer I (see recipe)
  • Substrate solution I (see recipe)
  • Gradient fractions (see protocol 1 or protocol 52 or protocol 2 or protocol 32)
  • 0.25 M NaOH

Support Protocol 2: Assay for β‐N‐Acetylglucosaminidase

  Materials
  • Assay buffer II (see recipe)
  • Substrate solution II (see recipe)
  • Bicarbonate buffer (see recipe)
  • 1.2 mM (21 mg/100 ml) 4‐methylumbelliferone, prepare fresh
  • Fluorimeter and appropriate cuvettes
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Figures

Videos

Literature Cited

Literature Cited
   Akasaki, K., Fukuzawa, M., Kinoshita, H., Furuno, K., and Tsuji, H. 1993. Cycling of two endogenous lysosomal membrane proteins, lamp‐2 and acid phosphatase, between the cell surface and lysosomes in cultured rat hepatocytes. J. Biochem. 114:598‐604.
   Arai, K., Kanaseki, T., and Ohkuma, S. 1991. Isolation of highly purified lysosomes from rat liver: Identification of electron carrier components on lysosomal membranes. J. Biochem. 110:541‐547.
   Balch, W.E. and Rothman, J.E. 1985. Characterization of protein transport between successive compartments of the Golgi apparatus: Asymmetric properties of donor and acceptor activities in cell‐free systems. Arch. Biochem. Biophys 240:413‐425.
   Barrett, A.J. and Heath, M.F. 1977. Lysosomal enzymes. In Lysosomes—A Laboratory Manual. 2nd. ed. (J.T. Dingle, ed.) pp. 19‐145. North Holland Publishing Co., Amsterdam.
   Berg, T., Kindberg, G.M., Ford, T., and Blomhoff, R. 1985. Intracellular transport of asialoglycoproteins in rat hepatocytes. Evidence for two subpopulations of lysosomes. Exp. Cell Res. 161:285‐296.
   Bridges, K.R. 1987. Ascorbic acid inhibits lysosomal autophagy of ferritin. J. Biol. Chem. 262:14773‐14778.
   Caimi, L., Marchesini, S., Aleo, M.F., Bresciani, R., Monti, E., Casella, A., Giudici, M.L., and Preti, A. 1989. Rapid preparation of a distinct lysosomal population from myelinating mouse brain using Percoll gradients. J. Neurobiol. 52:1722‐1728.
   Carlsson, S.R. and Fukuda, M. 1992. The lysosomal membrane glycoprotein Lamp‐1 is transported to lysosomes by two alternative pathways. Arch. Biochem. Biophys. 296:630‐639.
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   Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J.W., Fowler, S., and de Duve, C. 1968. The large‐scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with Triton WR 1339. J. Cell Biol. 37:482‐513.
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   Muno, D., Sutoh, N., Watanabe, T., Uchiyama, Y., and Kominami, F. 1990. Effect of metabolic alterations on the density and the contents of cathepsins B, H, and L of lysosomes in rat macrophages. Eur. J. Biochem. 191:91‐98.
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   Olsson, G.M., Svensson, I., Zdoisek, J.M., and Brunk, U.T. 1989. Lysosomal enzyme leakage during hypoxanthine/xanthine oxidase reaction. Virchows Arch. B Cell Pathol. 56:385‐391.
   Osmundsen, H. 1982. Factors which can influence β‐oxidation by peroxisomes isolated from livers of clofibrate‐treated rats. Some properties of peroxisomal fractions isolated in a self‐generated Percoll gradient by vertical rotor centrifugation. Int. J. Biochem. 14:905‐914.
   Pisoni, R.L., Flickinger, K.S., Thoene, J.G., and Christensen, H.N. 1987. Characterization of carrier‐mediated transport systems for small neutral amino acids in human fibroblast lysosomes. J. Biol. Chem. 262:610‐617.
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   Yamaguchi, T. and Kaneda, M. 1988. Presence of cytochrome b‐558 in guinea pig alveolar macrophages‐subcellular localization and relationship with NADPH oxidase. Biochim. Biophys. Acta 933:450‐459.
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