Improve Research Reproducibility A Bio-protocol resource

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往期刊物

Intact in situ Preparation of the Drosophila melanogaster Lymph Gland for a Comprehensive Analysis of Larval Hematopoiesis

黑腹果蝇淋巴腺的完整原位制备用于综合幼虫造血分析
DR Diana Rodrigues
KV K. VijayRaghavan
LW Lucas Waltzer
MI Maneesha S. Inamdar
Q&A 0 Q&A
4682 Views
Nov 5, 2021

Blood cells have a limited lifespan and are replenished by a small number of hematopoietic stem and progenitor cells (HSPCs). Adult vertebrate hematopoiesis occurs in the bone marrow, liver, and spleen, rendering a comprehensive analysis of the entire HSPC pool nearly impossible. The Drosophila blood system is well studied and has developmental, molecular, and functional parallels with that of vertebrates. Unlike vertebrates, post-embryonic hematopoiesis in Drosophila is essentially restricted to the larval lymph gland (LG), a multi-lobed organ that flanks the dorsal vessel. Because the anterior-most or primary lobes of the LG are easy to dissect out, their cellular and molecular characteristics have been studied in considerable detail. The 2-3 pairs of posterior lobes are more delicate and fragile and have largely been ignored. However, posterior lobes harbor a significant blood progenitor pool, and several hematopoietic mutants show differences in phenotype between the anterior and posterior lobes. Hence, a comprehensive analysis of the LG is important for a thorough understanding of Drosophila hematopoiesis. Most studies focus on isolating the primary lobes by methods that generally dislodge and damage other lobes. To obtain preparations of the whole LG, including intact posterior lobes, here we provide a detailed protocol for larval fillet dissection. This allows accessing and analyzing complete LG lobes, along with dorsal vessel and pericardial cells. We demonstrate that tissue architecture and integrity is maintained and provide methods for quantitative analysis. This protocol can be used to quickly and effectively isolate complete LGs from first instar larval to pupal stages and can be implemented with ease.

Hypochlorous Acid Staining with R19-S in the Drosophila Intestine upon Ingestion of Opportunistic Bacteria

果蝇肠道摄入外来细菌后次氯酸R19-S染色
SH Salma Hachfi
OB Olivia Benguettat
AG Armel Gallet
Q&A 0 Q&A
6587 Views
May 20, 2019
The intestine is endowed with an innate immune system that is required to fight any exogenous bacteria that are swallowed along with the food. The first line of defense that is mounted by the gut epithelium is the release of immune Reactive Oxygen Species (ROS), such as hypochlorous acid (HOCl), into the lumen. HOCl is produced within 1.5 h of bacterial ingestion and is very labile once released. Therefore, to monitor HOCl production upon ingestion of allochthonous bacteria, one needs a detection system that can quickly and efficiently detect HOCl production in the intestine. While most of the ROS-sensitive probes available in the market detect all kinds of ROS without any distinction, the R19-S fluorescent probe has been developed to specifically detect HOCl. Here, we describe a protocol to monitor HOCl production using this probe in the gut lumen of adult Drosophila upon ingestion of the opportunistic bacteria Bacillus thuringiensis.

Drosophila Model of Leishmania amazonensis Infection

亚马逊利什曼原虫感染的果蝇模型
KO Kendi Okuda
NS Neal Silverman
Q&A 0 Q&A
7800 Views
Dec 5, 2017
This protocol describes how to generate and harvest antibody-free L. amazonensis amastigotes, and how to infect adult Drosophila melanogaster with these parasites. This model recapitulates key aspects of the interactions between Leishmania amastigotes and animal phagocytes.

Isolation and Infection of Drosophila Primary Hemocytes

果蝇原代血细胞的分离和感染
CT Charles Tracy
HK Helmut Krämer
Q&A 1 Q&A
11335 Views
Jun 5, 2017
Phagocytosis of invading pathogens and their subsequent clearance in lysosomes is important for organismal fitness. We have devised the following protocol to extract phagocytic hemocytes from wild-type and mutant Drosophila larvae and infect the isolated hemocytes with GFP-labeled E. coli to measure the rate of phagocytosis and degradation within individual hemocytes over time.

Escherichia coli Infection of Drosophila

果蝇的大肠埃希杆菌感染
CT Charles Tracy
HK Helmut Krämer
Q&A 0 Q&A
9557 Views
May 5, 2017
Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The following protocol is designed to reproducibly inject a known amount of non-pathogenic E. coli into otherwise sterile flies and to measure the survival of flies after infection. The protocol can be easily adapted to different types of bacteria.