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Immunology and Infection
Stworzenie systemu in vitro do badania wewnątrzkomórkowego zachowania Candida glabra...
Stworzenie systemu in vitro do badania wewnątrzkomórkowego zachowania Candida glabra...
JoVE Journal
Immunology and Infection
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JoVE Journal Immunology and Infection
Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Stworzenie systemu in vitro do badania wewnątrzkomórkowego zachowania Candida glabrata w ludzkich makrofagach THP-1

Full Text
12,263 Views
13:47 min
December 10, 2013

DOI: 10.3791/50625-v

Maruti Nandan Rai1, Sapan Borah1, Gaurav Bairwa*1, Sriram Balusu*1,2, Neelima Gorityala1, Rupinder Kaur1

1Laboratory of Fungal Pathogenesis,Centre for DNA Fingerprinting and Diagnostics, Andhra Pradesh, India, 2Current location: VIB Department for Molecular Biomedical Research, UGent,Fiers-Schell-Van Montagu Building, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium

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Overview

This article presents a protocol for establishing an in vitro cell culture model to investigate the interaction between the human fungal pathogen Candida glabrata and human macrophages. This model aims to enhance understanding of fungal virulence mechanisms.

Key Study Components

Area of Science

  • Microbiology
  • Immunology
  • Cell Biology

Background

  • Candida glabrata is a facultative intracellular pathogen.
  • Understanding its interaction with macrophages is crucial for insights into fungal infections.
  • THP-1 cell line is commonly used for macrophage differentiation.
  • In vitro models are essential for studying pathogen-host interactions.

Purpose of Study

  • To develop a reliable in vitro model for studying Candida glabrata.
  • To assess the intracellular behavior of the pathogen in human macrophages.
  • To evaluate macrophage responses to Candida infection.

Methods Used

  • THP-1 monocytes are differentiated into macrophages using PMA treatment.
  • Macrophages are infected with Candida glabrata cells.
  • Non-phagocytosed cells are removed through PBS washes.
  • Intracellular cells are recovered for further analysis.

Main Results

  • The protocol allows for the assessment of Candida glabrata virulence in macrophages.
  • It enables the study of cytokine responses in THP-1 macrophages.
  • Insights into the maturation processes of macrophages upon infection are provided.

Conclusions

  • This in vitro model is a valuable tool for studying fungal pathogenesis.
  • It contributes to the understanding of host-pathogen interactions.
  • Future studies can leverage this model to explore therapeutic interventions.

Frequently Asked Questions

What is the significance of studying Candida glabrata?
Candida glabrata is an opportunistic pathogen that poses a significant threat to immunocompromised individuals, making its study crucial for developing effective treatments.
How are THP-1 cells differentiated into macrophages?
THP-1 cells are treated with PMA to induce differentiation into macrophage-like cells.
What techniques are used to analyze intracellular Candida glabrata?
The protocol includes washing non-phagocytosed cells and recovering intracellular cells for further analysis.
What are the expected outcomes of this study?
The study aims to elucidate the virulence mechanisms of Candida glabrata and the immune response of macrophages.
Can this model be used for other pathogens?
Yes, the model can potentially be adapted to study other intracellular pathogens.
What is the role of cytokines in this study?
Cytokines are key indicators of the immune response and are measured to assess macrophage activation during infection.

Obecny artykuł przedstawia protokół do ustanowienia systemu modelowego hodowli komórkowych in vitro do badania interakcji fakultatywnego wewnątrzkomórkowego patogenu grzyba ludzkiego Candida glabrata z ludzkimi makrofagami, co będzie użytecznym narzędziem do poszerzenia naszej wiedzy o mechanizmach wirulencji grzybów.

Ogólnym celem tej procedury jest ustanowienie systemu modelowego hodowli in vitro w celu zbadania wewnątrzkomórkowego zachowania ludzkiego oportunistycznego patogenu grzybowego. Canida lab ratta w makrofagach pochodzących z ludzkiej linii komórek monocytowych TP one. Aby osiągnąć ten THP, jeden monocyt jest najpierw traktowany czterema estrami, aby zróżnicować je w makrofagi.

Następnie makrofagi THP one są zakażane komórkami GTA canida dwie godziny po zakażeniu. Komórki GTA niefagocytozy canida są usuwane przez myjki PBS, a wewnątrzkomórkowe komórki e są odzyskiwane do dalszych badań przez osmos zakażonych makrofagów. Protokół ten może być wykorzystany do oceny zarówno komórek GTA wirusa do canida w makrofagach, jak i dojrzałości Rossa faiza i odpowiedzi cytokin w THP na makro FIDS po zakażeniu Candida GTA.

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