Name: 인간 혈액에서 말초 혈액 단핵 세포(PBMC)의 분리 및 식세포작용 분석을 위한 대식세포로의 PBMC의 분화
Uploaded: 2023-10-20T13:10:00
Description: Watch this Scientific Journal Video about Isolation of Peripheral Blood Mononuclear Cells PBMCs from Human Blood and Differentiation of PBMCs into Macrophages for Phagocytosis Assay at JoVE.com

isolation of peripheral blood mononuclear cells (pbmcs) from human blood and differentiation of pbmcs into macrophages for phagocytosis assay

Fungal Clearance Efficiency of Macrophages

The Trichoderma genus (Order: Hypocreales, Family: Hypocreaceae) is composed of ubiquitous, saprophytic fungi that are parasites of other fungal species and are capable of producing a range of commercially useful enzymes1. These fungal species are used for the production of heterologous proteins2, the production of cellulose3, ethanol, beer, wine, and paper4, in the textile industry5, food industry6, and in agriculture as biological control agents7,8. In addition to the industrial interest in these fungal species, the increasing number of infections in humans has given some Trichoderma species the status of opportunistic pathogens9.
Trichoderma spp. grow rapidly in culture, with initially white and cottony colonies that turn greenish yellow to dark green10. They are adapted to live in a wide range of pH and temperature conditions, and the opportunistic species are able to survive at physiological pH and temperatures and, thus, colonize different human tissues11,12,13. Importantly, the rise in the infection rate of Trichoderma spp. may be associated with virulence factors, and these are not well studied. In addition, studies focusing on understanding the immune response against opportunistic Trichoderma species are still rare.
During an infection, along with neutrophils, macrophages represent the line of defense responsible for phagocytosis, and, thus, prevent the growth and colonization of pathogens in different tissues. Using pattern recognition receptors, such as Toll-like receptors and C-type lectin receptors, macrophages phagocytose fungi and process them into phagolysosomes, thus promoting a respiratory burst, the release of pro-inflammatory cytokines, and the destruction of the phagocytosed microorganisms14. The mechanism of phagocytosis, however, can be affected and evaded by different microbial strategies, such as the size and shape of the fungal cells; the presence of capsules that hinder phagocytosis; decreasing the number of phagocytosis-inducing receptors; the remodeling of the structure of actin fibers in the cytoplasm; hindering the formation of pseudopodia; and phagosome or phagolysosome escape after the phagocytosis process14.
Many pathogens, including Cryptococcus neoformans, use macrophages as a niche to survive in the host, disseminate, and induce infection15. The phagocytosis and clearance assay is used to evaluate the immune response against pathogens and to identify the microbial strategies employed to evade the innate immune system15,16,17. This type of technique can also be used to examine the differential kinetics of phagocytosis, delayed phagosome acidification, and oxidative burst that result in reduced fungal killing18.
Different methods can be used to evaluate phagocytosis, fungal survival, and the evasion of the phagosome maturation process. These include fluorescence microscopy, which is used to observe phagocytosis, the cellular location, and the molecules produced during phagocytosis19; flow cytometry, which provides quantitative data on phagocytosis and is used to evaluate the different markers involved in the process20,21; intravital microscopy, which is used to assess microbial capture and phagosome maturation22; antibody-mediated phagocytosis, which is used to assess the specificity of the phagocytosis process for a pathogen23; and others24,25,26,27.
The protocol presented here employs a common, low-cost, and direct method using an optical microscope and plate growth assay to assess the phagocytosis and killing of fungal conidia. This protocol will provide the readers with step-by-step instructions for performing the phagocytosis and clearance assay using human peripheral blood mononuclear-derived macrophages exposed to T. stromaticum. PBMCs were used because Trichoderma conidia are applied as a biocontrol against phytopathogens and a biofertilizer for plant crops worldwide and have caused several human infections, called Trichodermosis. Besides that, there are only two previous works focusing on the interaction between Trichoderma conidia and the human immune system, in which we examined neutrophils28 and autophagy in macrophages29. This article shows first how the phagocytosis of the conidia of T. stromaticum by PBMC-derived macrophages can be studied, and then how the viability of the engulfed conidia can be assessed using simple microscopy-based techniques. This protocol may further facilitate investigations on macrophage-associated immune response or immune system modulation-related mechanisms.

Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System

Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages의 생존력 분석

Watch this Scientific Journal Video about Isolation of Peripheral Blood Mononuclear Cells PBMCs from Human Blood and Differentiation of PBMCs into Macrophages for Phagocytosis Assay at JoVE.com

인간 혈액에서 말초 혈액 단핵 세포(PBMC)의 분리 및 식세포작용 분석을 위한 대식세포로의 PBMC의 분화

기증자로부터 채취한 혈액을 50밀리리터 튜브로 옮기고 PBS를 추가하여 최종 부피 35밀리리터를 얻습니다. 폴리자당 용액이 들어 있는 튜브의 벽을 따라 혈액을 천천히 옮겨 바이페이직 현탁액을 형성합니다. 즉시, 섭씨 24도에서 30분 동안 1, 600G에서 현탁액을 원심분리합니다.원심분리 후 PBMC를 포함하는 흰색 고리의 형성을 관찰하고 15밀리리터 튜브에서 부드럽게 흡인하여 수집합니다. 그런 다음 PBMC가 들어 있는 튜브에 PBS를 추가하여 최종 부피 10밀리리터를 얻습니다. 섭씨 24도에서 30분 동안 1, 600G의 원심분리기에 현탁액을 균질화합니다.원심분리 후에, PBS의 10 밀리리터를 가진 5 분 동안 1, 600 G에 세포를 3 시간 세척하십시오. R10 배지 2밀리리터에 펠릿을 재현탁하기 전에.

Research

JoVE Journal

Immunology and Infection

Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Blood and Differentiation of PBMCs into Macrophages for Phagocytosis Assay

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial ...

연구

면역학 및 감염병학

Culturing Trichoderma stromaticum and Obtaining Conidia for Phagocytosis Assay

Trichoderma stromaticum 배양 및 식세포작용 분석을 위한 분생포자 확보

Phagocytosis Kinetics and Trichoderma stromaticum Conidial Viability Assay Using Human Peripheral Blood Mononuclear-Derived Macrophages

Phagocytosis Kinetics 및 Trichoderma stromaticum Conidial Viability Assay Using Human Peripheral Blood Mononuclear-Derived Macrophages(인간 말초 혈액 단핵 유래 대식세포를 사용한 식세포작용 역학 및 Trichoderma stromaticum Conidial Viability Assay)