Name: Ensaios de fluorescência para o estudo da interação do Mycobacterium tuberculosis com o receptor imunológico SLAMF1 (Vídeo) | JoVE Journal
Uploaded: 2025-02-28T14:00:00.000+00:00
Duration: 7 min 42 s
Description: Aprenda ensaios de fluorescência para estudar a interação do Mycobacterium tuberculosis com o receptor SLAMF1 em macrófagos humanos. Este protocolo utiliza citometria de fluxo e microscopia para investigar respostas imunológicas.

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</ol>

<table><tbody><tr><td>Anticorpo secundário Alexa Fluor 488</td><td>Invitrogen</td><td>A21121</td><td>Para microscopia de fluorescência</td></tr><tr><td>anticorpo anti-SLAMF1 FITC&nbsp;</td><td>eBiociência</td><td>11-1509-42</td><td>Para citometria de fluxo</td></tr><tr><td>anticorpo anti-SLAMF1 PE&nbsp;</td><td>BioLegend</td><td>306308</td><td>Para citometria de fluxo</td></tr><tr><td>anticorpo primário anti-SLAMF1</td><td>BioLegend</td><td>306302</td><td>Para microscopia de fluorescência</td></tr><tr><td>Aqua-Poly/Montagem</td><td>Policiências</td><td>18606-20</td><td>Mídia de montagem</td></tr><tr><td>Microesferas CD14</td><td>Miltenyi Biotec</td><td>130-097-052</td><td>Para isolamento de monócitos</td></tr><tr><td>Lamínulas 12mm</td><td>HDA</td><td>-</td><td>Para ensaio de interação por microscopia</td></tr><tr><td>EGS</td><td>ThermoFisher Scientific&nbsp;</td><td>21565</td><td>Para tratamento de reticulação</td></tr><tr><td>FACSCanto II</td><td>BD Biociências</td><td>338960</td><td>Citômetro de fluxo com BD FACSDiva software</td></tr><tr><td>Soro Fetal Bovino</td><td>Natocor</td><td>-</td><td>Inativado e irradiado, para cultura de macrófagos</td></tr><tr><td>Ficoll-Paque PLUS</td><td>Cytiva</td><td>17144003</td><td>Para separação de PBMCs</td></tr><tr><td>Fiji/ImageJ</td><td>Software de código aberto</td><td>-</td><td>Para análise de micrografias</td></tr><tr><td>FlowJo 7.6.2&nbsp;</td><td>Estrela da Árvore</td><td>-</td><td>Para análise de citometria de fluxo</td></tr><tr><td>Formaldeído</td><td>Merck</td><td>K47740803613</td><td>Para tratamento de reticulação</td></tr><tr><td>Lâminas de vidro</td><td>Vidro Klass</td><td>-</td><td>Para ensaio de interação por microscopia</td></tr><tr><td>Glicina</td><td>Sigma</td><td>G8898</td><td>Para tratamento de reticulação</td></tr><tr><td>Imager.A2</td><td>Carl Zeiss</td><td>430005-9901-000</td><td>Microscópio de fluorescência com módulo de iluminação Colibri 7</td></tr><tr><td>iMark</td><td>BIO-RAD</td><td>1681130</td><td>Leitor de absorbância de microplacas&nbsp;</td></tr><tr><td>L-glutamina</td><td>Sigma Aldrich</td><td>49419</td><td>Para cultura de macrófagos</td></tr><tr><td>M. tuberculosis, estirpe H37Rv, células inteiras irradiadas com raios gama</td><td>Recursos BEI, NIAID, NIH</td><td>NR-14819</td><td>Para ensaio de interação</td></tr><tr><td>M. tuberculosis, estirpe H37Rv, lisado de células inteiras</td><td>Recursos BEI, NIAID, NIH</td><td>NR-14822</td><td>Para estimulação de macrófagos e ensaio de interação</td></tr><tr><td>Neofuga 13R</td><td>Força de Cura</td><td>Neofuga 13R</td><td>Centrífuga refrigerada de alta velocidade para extração de proteínas</td></tr><tr><td>Penicilina/estreptomicina</td><td>Gibco</td><td>15140122</td><td>Para cultura de macrófagos</td></tr><tr><td>PMSF</td><td>ThermoFisher Scientific</td><td>36978</td><td>Para isolamento de proteínas</td></tr><tr><td>Poli-D-Lisina</td><td>Sigma Aldrich</td><td>A-003-M</td><td>Para tratamento de lamínulas</td></tr><tr><td>Coquetel de inibidores de protease</td><td>Sigma Aldrich&nbsp;</td><td>Pág. 8340</td><td>Para isolamento de proteínas</td></tr><tr><td>Rodamina B&nbsp;</td><td>Sigma Aldrich&nbsp;</td><td>21955</td><td>Para coloração de M. tuberculosis</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875093</td><td>Para cultura de macrófagos</td></tr><tr><td>Centrífuga Sorvall ST 16/16R</td><td>ThermoFisher Scientific</td><td>75004240</td><td>Para isolamento de PBMCs e monócitos</td></tr></tbody></table>

ensaios de fluorescência para o estudo da interação do mycobacterium tuberculosis com o receptor imunológico slamf1

Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1

Neste trabalho, é fornecido um protocolo que permite a avaliação da interação de M. tuberculosis com o receptor imune SLAMF1 em macrófagos humanos (Figura 1). Para tanto, foi obtido sangue periférico de doadores saudáveis. Em seguida, as PBMCs foram separadas por centrifugação sobre o meio de gradiente de densidade e os monócitos foram isolados por seleção magnética positiva (≥95% de pureza, Figura 2A,B). Os monócitos isolados foram ade...

Ensaios de fluorescência para o estudo da interação do Mycobacterium tuberculosis com o receptor imunológico SLAMF1

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic ...

fluorescence-assays-for-study-mycobacterium-tuberculosis-interaction

Research

JoVE Journal

Immunology and Infection

Imunologia e Infecção

Fonte: Barbero, A. M., et.al. Ensaios de fluorescência para o estudo da interação entre Mycobacterium tuberculosis e o receptor imunológico SLAMF1. J. Vis. Exp. ( (2025)Este vídeo demonstra um ensaio baseado em fluorescência para estudar a interação entre um receptor de macrófagos e antígenos do Mycobacterium tuberculosis . Antígenos marcados fluorescentemente em um coverslip são incubados com extratos de proteína de macrófagos, reticulados, marcados por anticorpos e visualizados por sinais de...

Ensaio baseado em fluorescência para investigar interações entre receptores de macrófagos e antígenos bacterianos

Source: Barbero, A. M., et al. Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1. J. Vis. Exp. (2025)This video demonstrates the procedure for labeling host immune receptors that bind bacterial surface antigens, enabling their detection through flow cytometry.

Analysis of Macrophage Receptor Interactions with Pathogenic Bacteria Using Flow Cytometry

Este vídeo demonstra a imagem de fluorescência enzimática repórter (REF) para detecção in vivo da infecção por Mycobacterium tuberculosis. Os camundongos infectados são injetados intraperitonealmente com um substrato para imagens REF. Ao ser hidrolisado pela enzima β-lactamase bacteriana, o substrato emite fluorescência, cuja detecção auxilia na avaliação da infecção.

Imagem de fluorescência enzimática repórter para rastreamento in vivo de infecção bacteriana

Descreve-se um método para controlar a ruptura endomembranar provocada pelas bactérias...

Medidas de ruptura vacuolar única célula causadas por patógenos intracelulares

Aqui, descrevemos um ensaio fenotípico aplicável às telas de alto rendimento/alto conteúdo de pequenas interfissões de RNA sintético (siRNA), composto químico e bibliotecas mutantes mycobacterium tuberculosis. Este método baseia-se na detecção de Mycobacterium tuberculosis rotulado fluorescentemente dentro de célula hospedeira fluorescente rotulada usando microscopia confocal automatizada.

Um ensaio fenotípico microscópico para quantificação de micobactérias intracelulares adaptadas para triagem de alto rendimento/alto conteúdo

Nós desenvolvemos um sistema de rastreio de alto rendimento modular para descobrir novos compostos contra o Mycobacterium tuberculosis, direccionamento intracelular e em caldo de crescimento de bactérias, bem como a citotoxicidade contra a célula hospedeira de mamífero.

Sistema de Eficácia e citotoxicidade Rastreio de inibidores Segmentação intracelular<em&gt; Mycobacterium tuberculosis</em

Novos modelos e ensaios que melhorariam o processo inicial de desenvolvimento de medicamentos para a próxima geração de medicamentos antituberculose são altamente desejáveis. Aqui, descrevemos um ensaio rápido, barato e compatível com BSL-2 para avaliar a eficácia do medicamento contra Mycobacterium tuberculosis que pode ser facilmente adaptado para triagem de alto rendimento.

Um ensaio Compatível de alta capacidade para avaliar droga Eficácia contra macrófagos passadas<em&gt; Mycobacterium tuberculosis</em

Descrevemos a imagem óptica de camundongos infectados com Mycobacterium tuberculosis (M. tuberculosis) usando fluorescência de enzima repórter (REF). Esse protocolo proporciona uma detecção sensível e específica de M. tuberculosis em modelos animais pré-clínicos para investigação patogenia, terapêutica e vacina.

Mycobacterium tuberculosis em camundongos com fluorescência de enzima do repórter de imagem

Este estudo desenvolveu um ensaio para triagem de alto rendimento contra Mycobacterium abscessus com uma cepa repórter dupla recém-criada, usando fluorescência e luminescência para avaliar a viabilidade bacteriana rapidamente. Este protocolo será relevante para pesquisadores que pretendem rastrear novos medicamentos contra essa bactéria resistente a medicamentos.

Desenvolvimento de ensaio para triagem de medicamentos de alto rendimento contra micobactérias

Aqui, apresentamos um protocolo para imagem intravital de um Mycobacterium tuberculosis aprovado por BSL-2 atenuado. Cepa no pulmão murino usando uma janela pulmonar permanente. Essa abordagem permite a análise longitudinal com resolução de célula única das interações precoces hospedeiro-patógeno, incluindo localização bacilar, dinâmica vascular e captação de macrófagos dentro do mesmo microambiente pulmonar.

Imagem intravital de Mycobacterium tuberculosis auxotrófico atenuado no pulmão murino durante infecção intravascular precoce

Aprenda ensaios de fluorescência para estudar a interação do Mycobacterium tuberculosis com o receptor SLAMF1 em macrófagos humanos. Este protocolo utiliza citometria de fluxo e microscopia para investigar respostas imunológicas.

Ensaios de fluorescência para o estudo da interação do Mycobacterium tuberculosis com o receptor imunológico SLAMF1

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