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Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays

Featured protocol,  Authors: Ashwin D’Souza
Ashwin D’SouzaAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3850
Paulomi Sanghavi
Paulomi SanghaviAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3851
Ashim Rai
Ashim RaiAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3852
Divya Pathak
Divya PathakAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3853
 and Roop Mallik
Roop MallikAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
For correspondence: roop@tifr.res.in
Bio-protocol author page: a3854
date: 12/5/2016, 73 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2056.

Brief version appeared in Cell, Feb 2016
We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation and pathogen clearance.

Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays

Authors: Ashwin D’Souza
Ashwin D’SouzaAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3850
Paulomi Sanghavi
Paulomi SanghaviAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3851
Ashim Rai
Ashim RaiAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3852
Divya Pathak
Divya PathakAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3853
 and Roop Mallik
Roop MallikAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
For correspondence: roop@tifr.res.in
Bio-protocol author page: a3854
date: 12/5/2016, 73 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2056.

[Abstract] We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to ...

In vitro Fluorescent Matrix Degradation Assay for Entamoeba histolytica

Authors: Merlyn Emmanuel
Merlyn EmmanuelAffiliation: Indian Institute of Science Education and Research Bhopal, Bhopal, India
Bio-protocol author page: a3003
 and Sunando Datta
Sunando DattaAffiliation: Indian Institute of Science Education and Research Bhopal, Bhopal, India
For correspondence: sunando@iiserb.ac.in
Bio-protocol author page: a3004
date: 3/20/2016, 682 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1769.

[Abstract] Fluorescent matrix degradation assay is a popular and widely used assay in the field of invadopodium biology (Artym et al., 2009). Matrix remodeling and degradation can be observed under both physiological and pathological conditions. Cancer cells extensively remodel and degrade the underlying matrix ...

Dictyostelium Cultivation, Transfection, Microscopy and Fractionation

Authors: Jennifer Hirst
Jennifer HirstAffiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK
For correspondence: jh228@cam.ac.uk
Bio-protocol author page: a2250
Robert R Kay
Robert R KayAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
Bio-protocol author page: a2251
 and David Traynor
David TraynorAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
For correspondence: dt101@mrc-lmb.cam.ac.uk
Bio-protocol author page: a2252
date: 6/5/2015, 1893 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1485.

[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. ...

Cyst Detection in Toxoplasma gondii Infected Mice and Rats Brain

Authors: Valeria Bellini
Valeria BelliniAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
Bio-protocol author page: a2111
Corinne Loeuillet
Corinne LoeuilletAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
Bio-protocol author page: a2112
Céline Massera
Céline MasseraAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
Bio-protocol author page: a2113
Marie-France Cesbron-Delauw
Marie-France Cesbron-DelauwAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
For correspondence: marie-france.cesbron@ujf-grenoble.fr
Bio-protocol author page: a2114
 and Pierre Cavaillès
Pierre CavaillèsAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
For correspondence: pierre.cavailles@ujf-grenoble.fr
Bio-protocol author page: a2115
date: 4/5/2015, 1646 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1439.

[Abstract] Toxoplasmosis caused by the intracellular parasite Toxoplasma gondii, is characterized by a life-long chronic infection. The parasite is an efficient neurotropic infectious agent that establishes its “safe” life by forming intracellular cysts in chronically infected animals and humans. This protocol ...

A Gas Chromatography-Mass Spectrometry-Based Two Stage Assay for Measurement of in vitro myo-Inositol 3-phosphate Synthase (INO1) Activity

Authors: James I. MacRae
James I. MacRaeAffiliation 1: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia
Affiliation 2: The National Institute for Medical Research, The Ridgeway, London, UK
For correspondence: jmacrae@nimr.mrc.ac.uk
Bio-protocol author page: a2049
 and Malcolm J. McConville
Malcolm J. McConvilleAffiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia
Bio-protocol author page: a2050
date: 3/5/2015, 1887 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1418.

[Abstract] This method describes an in vitro assay for measuring INO1 enzyme activity (the conversion of glucose 6-phosphate to myo-inositol 3-phosphate) in cell-free extracts. The method was first described for Plasmodium falciparum cells in MacRae et al. (2014) and consists of two parts: Part 1 describes the ...

Preparation of Parasite Protein Extracts and Western Blot Analysis

Authors: Arlett Heiber
Arlett Heiber Affiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1407
 and Tobias Spielmann
Tobias SpielmannAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
For correspondence: spielmann@bni-hamburg.de
Bio-protocol author page: a1408
date: 6/5/2014, 6240 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1136.

[Abstract] In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, ...

Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites

Authors: Arlett Heiber
Arlett HeiberAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1407
Silke Retzlaff
Silke RetzlaffAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1409
 and Tobias Spielmann
Tobias SpielmannAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
For correspondence: spielmann@bni-hamburg.de
Bio-protocol author page: a1408
date: 6/5/2014, 2045 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1137.

[Abstract] This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined ...

Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress

Authors: Fiona Hackett
Fiona HackettAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1182
Christine R Collins
Christine R CollinsAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1183
Malcolm Strath
Malcolm StrathAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1184
 and Michael J Blackman
Michael J BlackmanAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
For correspondence: mblackm@nimr.mrc.ac.uk
Bio-protocol author page: a1185
date: 3/5/2014, 2425 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1055.

[Abstract] Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the ...

Protein Sample Preparation for Proteomic Analysis in Leishmania donovani

Authors: Alexandros Alexandratos
Alexandros AlexandratosAffiliation: Molecular Parasitology Department, Hellenic Pasteur Institute, Athens, Greece
For correspondence: alexandratos@pasteur.gr
Bio-protocol author page: a1191
 and Despina Smirlis
Despina SmirlisAffiliation: Molecular Parasitology Department, Hellenic Pasteur Institute, Athens, Greece
Bio-protocol author page: a1192
date: 3/5/2014, 2415 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1058.

[Abstract] Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. These protozoa, regulate their gene expression in an atypical way, compared to other higher eukaryotes. The regulation of gene expression is characterized by a predominance of post-transcriptional ...

EdU labeling of Trypanosome Cells and Their Kinetoplast DNA (kDNA)

Author: Jianyang Wang
Jianyang WangAffiliation: Department of Cell Biology, Johns Hopkins University, Baltimore, MD, USA
For correspondence: jwang117@jhu.edu
Bio-protocol author page: a636
date: 6/20/2013, 2983 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.799.

[Abstract] Trypanosome mitochondrial genome, known as Kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings. The studies of kDNA replication and architecture are of major significance since kDNA is a valid drug target. However, DNA in procyclic trypanosomes can not be labeled with tracer concentrations ...
1 2 

Preparation of Parasite Protein Extracts and Western Blot Analysis

Authors: Arlett Heiber
Arlett Heiber Affiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1407
 and Tobias Spielmann
Tobias SpielmannAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
For correspondence: spielmann@bni-hamburg.de
Bio-protocol author page: a1408
date: 6/5/2014, 6240 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1136.

[Abstract] In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, ...

Plasmodium falciparum Rosette Formation Assay

Authors: Inès Vigan-Womas
Inès Vigan-WomasAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
For correspondence: ines.vigan-womas@pasteur.fr
Bio-protocol author page: a577
Micheline Guillotte
Micheline GuillotteAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
Bio-protocol author page: a576
 and Odile Mercereau-Puijalon
Odile Mercereau-PuijalonAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
Bio-protocol author page: a308
date: 4/20/2013, 4534 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.412.

[Abstract] Rosetting, i.e. the capacity of red blood cells (iRBCs) infected with mature parasite stages to bind two or more uninfected red blood cells (RBCs) is a virulence factor of Plasmodium falciparum. This protocol describes an in vitro assay to monitor rosette formation by P. falciparum-infected red blood ...

Determination of Toxoplasma gondii Replication in Naïve and Activated Macrophages

Authors: Emma Iaconetti*
Emma IaconettiAffiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA
Bio-protocol author page: a157
Brian Lynch*
Brian LynchAffiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA
Bio-protocol author page: a158
Nathaniel Kim
Nathaniel KimAffiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA
Bio-protocol author page: a159
 and Dana G. Mordue
Dana G. MordueAffiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA
For correspondence: dana_mordue@nymc.edu
Bio-protocol author page: a160
 (*contributed equally to this work) date: 11/20/2012, 4420 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.289.

[Abstract] Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the disease toxoplasmosis. Chronic infection is established through the formation of tissue cysts predominantly in cardiac and neurologic tissues. A defining characteristic of T. gondii is its ability to evade the host’s immune ...

Glycophosphosphingolipid (GSPL) Purification Protocol

Author: Tripti De
Tripti DeAffiliation: Division of Infectious Disease and Immunology, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India
For correspondence: triptide@iicb.res.in
Bio-protocol author page: a175
date: 12/5/2012, 3661 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.299.

[Abstract] Glycosylated ceramide phosphorylinositol are present in many species of fungi and mushrooms and bacteria and parasitic organisms like leishmania. These are usually membrane raft associated and are not easily extracted by conventional methodologies. This extraction method gives higher yield of the glycolipid. ...

Plasmodium falciparum Rosette Disruption Assay

Authors: Micheline Guillotte
Micheline GuillotteAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
For correspondence: micheline.guillotte-blisnick@pasteur.fr
Bio-protocol author page: a576
Odile Mercereau-Puijalon
Odile Mercereau-PuijalonAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
Bio-protocol author page: a308
 and Inès Vigan-Womas
Inès Vigan-WomasAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
Bio-protocol author page: a577
date: 4/20/2013, 3308 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.411.

[Abstract] Rosetting, i.e. the capacity of Plasmodium falciparum-infected red blood cells (iRBCs) to bind two or more uninfected red blood cells (RBCs) is associated with severe malaria in African children. Disruption of rosettes using small soluble inhibitors or specific antibodies is viewed as an interesting ...

EdU labeling of Trypanosome Cells and Their Kinetoplast DNA (kDNA)

Author: Jianyang Wang
Jianyang WangAffiliation: Department of Cell Biology, Johns Hopkins University, Baltimore, MD, USA
For correspondence: jwang117@jhu.edu
Bio-protocol author page: a636
date: 6/20/2013, 2983 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.799.

[Abstract] Trypanosome mitochondrial genome, known as Kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings. The studies of kDNA replication and architecture are of major significance since kDNA is a valid drug target. However, DNA in procyclic trypanosomes can not be labeled with tracer concentrations ...

Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress

Authors: Fiona Hackett
Fiona HackettAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1182
Christine R Collins
Christine R CollinsAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1183
Malcolm Strath
Malcolm StrathAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1184
 and Michael J Blackman
Michael J BlackmanAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
For correspondence: mblackm@nimr.mrc.ac.uk
Bio-protocol author page: a1185
date: 3/5/2014, 2425 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1055.

[Abstract] Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the ...

Protein Sample Preparation for Proteomic Analysis in Leishmania donovani

Authors: Alexandros Alexandratos
Alexandros AlexandratosAffiliation: Molecular Parasitology Department, Hellenic Pasteur Institute, Athens, Greece
For correspondence: alexandratos@pasteur.gr
Bio-protocol author page: a1191
 and Despina Smirlis
Despina SmirlisAffiliation: Molecular Parasitology Department, Hellenic Pasteur Institute, Athens, Greece
Bio-protocol author page: a1192
date: 3/5/2014, 2415 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1058.

[Abstract] Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. These protozoa, regulate their gene expression in an atypical way, compared to other higher eukaryotes. The regulation of gene expression is characterized by a predominance of post-transcriptional ...

Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites

Authors: Arlett Heiber
Arlett HeiberAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1407
Silke Retzlaff
Silke RetzlaffAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1409
 and Tobias Spielmann
Tobias SpielmannAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
For correspondence: spielmann@bni-hamburg.de
Bio-protocol author page: a1408
date: 6/5/2014, 2045 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1137.

[Abstract] This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined ...

Dictyostelium Cultivation, Transfection, Microscopy and Fractionation

Authors: Jennifer Hirst
Jennifer HirstAffiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK
For correspondence: jh228@cam.ac.uk
Bio-protocol author page: a2250
Robert R Kay
Robert R KayAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
Bio-protocol author page: a2251
 and David Traynor
David TraynorAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
For correspondence: dt101@mrc-lmb.cam.ac.uk
Bio-protocol author page: a2252
date: 6/5/2015, 1893 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1485.

[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. ...
1 2