在原初和活化的巨噬细胞中测定刚地弓形虫的复制

Emma  Iaconetti; Brian  Lynch; Nathaniel  Kim; Dana G.  Mordue

doi:10.21769/BioProtoc.289

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Peer-reviewed

Determination of Toxoplasma gondii Replication in Naïve and Activated Macrophages

在原初和活化的巨噬细胞中测定刚地弓形虫的复制

EI Emma Iaconetti ^*

BL Brian Lynch ^*

NK Nathaniel Kim

DM Dana G. Mordue email

(*contributed equally to this work) 发布: 2012年11月20日第2卷第22期 DOI: 10.21769/BioProtoc.289 浏览次数: 12758

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Cover of The Journal of Immunology, featuring study using the protocol.

Apr 2012

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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 defenses; specifically, T. gondii can invade and persist within host phagocytes, using them to disseminate to the brain and central nervous system where cysts are then formed. This protocol is used to evaluate the ability of Toxoplasma gondii to survive and replicate within naive and activated murine bone marrow-derived macrophages at the level of single infected cells. In the following protocol macrophages are naive or activated with IFN-γ and LPS but different activation stimuli can be utilized as well as different host cell populations and diverse inhibitors. Parasite replication is determined by evaluating the number of parasites per vacuole over time using immunofluorescence staining for parasties and microscopic analysis. Kinetic determination of parasite number per vacuole accurately reflects parasite replication over time as vacuoles-containing parasites do not fuse with one another. Isolation of murine bone marrow-derived macrophages, preparation of conditioned L929 cells for collection of macrophage colony-stimulating factor, and staining for fluorescence microscopy included in the protocol has broad applicability. This protocol works well for pathogens like Toxoplasma gondii that reside in vacuoles that do not fuse with one another and that can be visualized by microscopy.

Keywords: Toxoplasma (弓形虫)

Macrophages (巨噬细胞)

Cell-autonomous immunity (细胞自主免疫)

Nitric oxide (一氧化氮)

Materials and Reagents

Note: source of reagents is not critical to experiment.

Murine bone marrow derived macrophages (see protocol below)
Rabbit polyclonal antibody against Toxoplasma gondii (Fitzgerald Laboratories)
Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (H + L) (Life Technologies, Invitrogen™, catalog number: A-11012 )
Dulbecco’s modified ragle medium high glucose (DMEM) (1x) (Life Technologies, Invitrogen™, catalog number: 10313-039 )
Fetal calf serum (FCS) (Hyclone, catalog number: SH30396.03 ); heat inactivated 56 °C for 30 min
L-gluatamine (Hyclone, catalog number: SH30034.01 )
Penicillin/streptomycin (Hyclone, catalog number: SV30010 )
L929 cell (ATCC CCL1) conditioned media as a source of macrophage colony-stimulating factor (M-CSF) for maturation of bone marrow-derived macrophages (see protocol below)
Dulbeccos PBS (Ca²⁺ free and Mg²⁺ free) (DPBS) (Hyclone, catalog number: SH30028.02 )
Toxoplasma gondii parasites (tachyzoites), Tachyzoites are the haploid replicating stage of Toxoplasma gondii that can be grown in vitro in human fibroblast cells or other host cell types.
Lipopolysaccharide (LPS) (List Biological from E. Coli O55: B5 #203)
Recombinant murine IFN-γ (Pepro Tech, catalog number: 50813665 )
Aminoguanidine (Thermo Fisher Scientific, Acros Organics, catalog number: AC40078-1000 ) - inhibitor of inducible nitric oxide synthase
Sodium nitroprusside (SNP) (Thermo Fisher Scientific, catalog number: AC211640000 ) - nitric oxide donor
16% methanol-free paraformaldehyde EM grade (Electron Microscopy Sciences, catalog number: 15710 )
Triton X100 (Sigma-Aldrich, catalog number: T9284-500 )
Mounting media such as Vectashield containing DAPI to stain nucleus (Vector Laboratories, catalog number: H-1200 )
Cover slips for the chamber slides (22 x 50 x 1) (Thermo Fisher Scientific, catalog number: 12-548-5E )
4% paraformaldehyde
KCl
KH₂PO₄
NaCl
Na₂HPO₄
DPBS (we purchase it but the recipe is below) (see Recipes)
D10 media (see Recipes)
BMC media (see Recipes)
Blocking buffer (see Recipes)
Wash buffer (see Recipes)

Equipment

Fluorescence microscope with 100x phase or DIC objective
Tabletop centrifuge capable of holding 15 or 50 ml centrifuge tubes
Humidified CO₂ incubator at 37 °C
Bacteriological petri plates (100 mm x 15 mm) (Thermo Fisher Scientific, catalog number: 0875712 )
Zeiss inverted Axiovert 200
Hemocytometer
8-well chamber slides (BD Biosciences, catalog number: 354118 ) or cover slips inserted into wells in a 24 well plate
Sterile plastic 50 ml centrifuge tubes (brand is not important)

Procedure

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Iaconetti, E., Lynch, B., Kim, N. and Mordue, D. G. (2012). Determination of Toxoplasma gondii Replication in Naïve and Activated Macrophages. Bio-protocol 2(22): e289. DOI: 10.21769/BioProtoc.289.