This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.
Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.
The innate immune system, which consists of physical and chemical barriers to infection as well as cellular components, is evolutionary conserved across multicellular organisms1,2. As the first line of defense, the innate immune system plays a critical role in combating invading pathogens in all animals1,2,3. The components of the innate immune response include a wide range of cell types which are classified on the basis that they lack specificity and immunological memory2,3,4. In humans, these cell types include phagocytic monocytes and macrophages, neutrophils, and cytotoxic natural killer cells4,5. While having a functional immune system is imperative for host survival, it is clear that the function of immune cells declines with age, a phenomenon known as immunosenescence5,6. Being able to assess age related changes in the immune response, including different aspects of the process of phagocytosis, could aid in our understanding of immunosenescence. The procedure we describe here provides an effective and repeatable approach to evaluate and quantify phagocytic events by hemocytes in Drosophila melanogaster.
Drosophila is an ideal model for studying the immune response for many reasons. For one, there is an extensive set of genetic tools available that make it possible to easily manipulate gene expression in a tissue-dependent manner7. These tools include a collection of mutants, RNA interference stocks, GAL4/UAS stocks, and the Drosophila Genetic Reference Panel that contains 205 different inbred lines for which the entire genome sequences are catalogued8. The short life cycle of Drosophila and the large number of individuals produced allow researchers to test multiple individuals in a controlled environment, in a short period of time. This greatly improves the ability to identify subtle differences in immune responses to infection among genotypes, between sexes or across ages. Importantly, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals1,2.
In Drosophila, the process of phagocytosis that follows infection is carried out by phagocytic hemocytes called plasmatocytes, which are equivalent to mammalian macrophages9. Hemocytes are essential for recognizing a wide range of particles and clearing microbial pathogens9,10,11,12,13. These cells express a variety of receptors that must differentiate self from non-self, and initiate signaling events needed to carry out the phagocytic process10,11,12,13,14,15. Once a particle is bound, it starts to be internalized by reorganization of the actin cytoskeleton and remodeling of the plasma membrane to expand around the particle, forming a phagocytic cup11,12,13,14. During this process, another set of signals tells the cell to internalize the particle further by closing the phagocytic cup, forming a membrane-bound phagosome11,12,13,14,15. The phagosome then undergoes a maturation process, associating with different proteins and fusing with lysosomes, forming an acidic phagolysosome11,12,13,14,15. At this point, particles can efficiently be degraded and eliminated11,12,13,14,15. Drosophila studies have revealed that older flies (4 weeks of age) have a reduced ability to clear an infection compared to younger flies (1-week old), likely due, at least in part, to a decline in some aspects of phagocytosis16,17.
The method described here utilizes two separate fluorescently-labelled heat-killed E. coli particles, one bearing a standard fluorophore and one that is pH sensitive, to assess two different aspects of phagocytosis: the initial engulfment of particles, and the degradation of particles in the phagolysosome. In this assay, fluoro-particle fluorescence is observable when the particles are bound and engulfed by hemocytes, while pH sensitive particles fluoresce only in the low pH conditions of the phagosome. Fluorescent events can then be observed in hemocytes that localize along the dorsal vessel. We focus on hemocytes localized to the dorsal vessel, which provide an anatomical landmark to locate hemocytes that are known to contribute to bacterial clearance, and to consistently isolate them. However, hemocytes in other parts of the body and the hemolymph are also important for clearance. Although we have not studied this cell population, our general procedure could be applicable for phagocytic assays of these cells as well. One advantage of our approach is that we can quantify phagocytic events within individual hemocytes, allowing us to detect subtle variation in phagocytic processes. Other studies that visualize fluorescent events through the cuticle18,19 do not account for differences in the numbers of hemocytes present, which is especially important to consider in our case as total hemocyte counts are expected to change with age17.
1. Collect and age Drosophila
2. Prepare fluorescently labeled particles
3. Inject the flies
4. Dissecting the dorsal vessel
5. Fixation and staining
6. Mounting cuticles onto microscope slides and imaging
7. Analyze images
To illustrate the described injection methods, Figure 1A shows the injection site on Drosophila melanogaster, as well as how food dye allows for a visual confirmation that the fly was injected (Figure 1B). The addition of food dye also aids in the recognition of a clogged needle. Injections can be performed in the abdomen, but keep the injection site consistent across experiments. This will help minimize possible variations between each experiment.
To visualize the fluorescently labeled particles within resident hemocytes along the dorsal vessel, we dissected the dorsal vessel and attached abdominal cuticle. Figure 2A-F outlines the dissection methods.
To assess the age-specific ability of young and aged flies to carry out phagocytosis, hemocytes along the dorsal vessel are visualized using a fluorescent microscope. To ensure that only cells along the dorsal vessel are counted, antibodies or GFP-tagged genes for certain blood cell markers or heart specific collagen, such as Hemese and Hemolectin, or Pericardin (Figure 3), respectively, can be used22,23,24. Fluorescently labeled E. coli particles are 1 mm in length, while hemocytes are 10 mm in diameter17. Only those fluorescent events located within a 10 mm diameter centered on a DAPI-positive nucleus are counted (Figure 4). To quantify fluorescent events, ImageJ software is used (Figure 5).
Figure 1: Injection site and visual verification. (A) Lateral side of thorax is pierced with a pulled-capillary needle. (B) Injections are visually verified by adding green food dye to particle solution. Please click here to view a larger version of this figure.
Figure 2: Dorsal vessel dissection. (A) Pins are placed in the thorax and posterior abdomen (black arrows). (B-C) Two horizontal incisions (green arrows) are made at the posterior end of the abdomen (B), and anterior end (C). (D) A vertical incision (green arrow) is made down the middle of the abdomen, connecting the two horizontal cuts. (E) Optional pins (*) are used to filet open the abdominal cavity, exposing internal tissue. (F) Internal tissue (crop, gut, uterus, ovaries, fat bodies) is removed, exposing the dorsal vessel. Please click here to view a larger version of this figure.
Figure 3: Ventral view of a dissected dorsal vessel from a 5-week old female injected with pH sensitive particles, stained with antibody directed against Pericardin (A). Dotted white line outlines the lateral side of the dorsal vessel, with arrow pointing towards the anterior region. (B) Enlarged image of (A): clusters of hemocytes (blue arrow) that were actively degrading bacteria, within the first aortic chamber of the dorsal vessel. (C) Enlarged image of (A) outlining the extracellular matrix (ECM) collagen-like protein, Pericardin (green arrow), that holds the dorsal vessel in place24. Please click here to view a larger version of this figure.
Figure 4: Dissected dorsal vessel and associated hemocytes from a female fly injected with pH sensitive particles, or fluoro-particles. (A) The dorsal vessel and associated hemocytes with engulfed pH sensitive-labelled E. coli particles (red), or (E) fluoro-labelled E. coli particles (red), isolated from a 1-week old fly, after recovering for 60 min. (B,F) Magnified inset of (A) and (E) (white box), respectively, showing two individual hemocytes with countable events. (C) The dorsal vessel and associated hemocytes with engulfed pH sensitive-labelled E. coli particles(red), or ( G) fluoro-labelled E. coli particles (red), isolated from a 5-week old fly, after recovering for 60 min. (D,H) Magnified inset of (C) and (G) (white box), respectively, showing two individual hemocytes with countable events. Dotted white line outlines the lateral side of the dorsal vessel, with arrow pointing towards the anterior region. Nuclei stained with DAPI (blue). Please click here to view a larger version of this figure.
Figure 5: Quantifying phagocytic events within a 10 mm hemocytes using the cell counter in ImageJ. (A) After opening image(s) in ImageJ, the Cell Counter Notice tool can be used to keep track of phagocytic events per cell. (B) This tool will assign a different color to each cell selected to be counted, with each dot corresponding to a fluorescent event within that cell. Pressing ‘alt+y’ will display a table showing the number of events counted per cell. Please click here to view a larger version of this figure.
The protocol described here is a reliable way to quantify different aspects of phagocytosis, under controlled experimental conditions. We note that we have only tested this procedure with gram negative bacterial particles and results may differ if gram positive bacterial particles are used. Indeed, it would be interesting to compare the phagocytic responses to both gram negative and gram positive bacteria in different experimental conditions. The use of a nano-injector allows for precise control over injection volumes, ensuring each fly is injected with the same volume of particles. One limitation to the protocol comes from inconsistencies in particle preparations. Particles will aggregate once frozen, so small variations in dilution volumes, or lack of vortexing, can affect particle concentration between experiments. To minimize possible variations in particle concentrations between ages, it is beneficial to inject 1- and 5-week old flies on the same day, using the same needle and particle solution. Another potential drawback is that during dissections, the dorsal vessel and/or cuticle can easily be damaged if pins are not handled properly. To avoid disrupting the dorsal vessel, minimize the number of pins used per dissection. The advantage to this dissection method is that all fixation, washing and staining steps can be performed in the dissection plate. Because the cuticles are pinned down, this prevents the cuticles from being lost between steps.
Compared to existing methods18,19,25,26,27,28,29, the described protocol has its advantages and limitations. By dissecting the dorsal vessel, we are able to visualize and quantify individual hemocytes at this location. This makes it possible to detect subtle variations in phagocytic activity between experimental groups. Other methods visualize fluorescently labeled particles by collecting hemocytes using a Bleed/Scrape assay19,25,26,27, or through an intact ventral cuticle18,19,28,29; however, individual hemocytes cannot be assessed when visualized through the dorsal cuticle. The advantage of this protocol, when compared to the Bleed/Scrape method is that our method allows us to assess only those hemocytes associated with the dorsal vessel, and does account for circulating cells or those along the body wall, which may be functionally different. Dissecting the dorsal vessel also removes the need to include a second round of injections with a fluorescence quencher, like Trypan blue19,26. This is because any particles not bound to or engulfed by a cell will be washed away during wash steps. Conversely, alternative methods may be easier to perform because they do not require dissections. While dissecting the dorsal vessel is easy to learn, this step adds a level of complexity that may not be feasible in some experimental designs.
Although the described use of this in vivo phagocytosis assay is to assess and quantify phagocytic events between different ages, this protocol is highly adaptable and can be used to analyze different aspects of phagocytosis between genotype, sex, or tissue type. With phagocytosis being of central importance for most multicellular animals, understanding how this process declines with age could lead to better therapeutic treatments for the aging population. This approach offers long-term potential for elucidating aspects of age-related changes in the immune response, with special focus on phagocytosis.
The authors have nothing to disclose.
This work was supported by grants from the National Institutes of Health R03 AG061484-02 and the UMBC College of Natural and Mathematical Sciences Becton Dickinson Faculty Research Fund.
0.10 mm Insect pins | Fine Science Tools | 26002-10 | Here: pins are cut in half, and the sharp end is used |
1 mL sterile syringes | Becton Dickinson | 309602 | Filled with mineral oil to load needle |
15% Fetal Bovine Serum (FBS) | Gibco | 16000-044 | for dissection media |
16 % Paraformaldehyde | Electron Microscopy Sciences | 15710 | EM-grade, 4% working, diluted in 1X PBS |
1x Phosphate buffered saline (PBS) | Sigma | P3813 | |
3 mL Trasnfer Pipet | Falcon | 357524 | |
3.5" Glass Capillaries | Drummond | 3-000-203-G/X | 1.14mm O.D X 3.5" length X 0.53" I.D |
35×10 mm Petri dishes | Becton Dickinson | 351008 | Used as dissection plate, filled half way with Sylgard |
6x penicillin/streptomycin | Life Technologies | 15140-122 | for dissection media |
70% Glycerol | Sigma | G9012 | |
Analog Vortex mixer | VWR | 58816-121 | |
Biological point forceps, Dumont No. 5 | Fine Science Tools | 11295-10 | |
DAPI (4',6-diamidino-2-phenylindole) | Life Technologies | D1306 | Diluted 1:1000 in 1x PBST |
Drosophila strain | w[*]; P{w[+mC]=He-GAL4.Z}85, P{w[+mC]=UAS-GFP.nls}8 | ||
E. coli (K-12 strain) BioParticles™, Alexa Fluor™ 594 conjugate | Life Technologies | E23370 | |
Glass slides | Premiere | D17026102 | |
Live cell imaging solution | Life Technologies | A14291DJ | preferred buffer for particle preparation and dilutions |
Mineral oil | Mpbio | 194836 | |
Nanoject II automatic nanoliter injector | Drummond | 3-000-204 | |
Narrow Polystyrene Super Bulk Drosophila Vials | Genesee | 32-116SB | Size: 25 X 95 mm |
Nutating Mixer | Fisher Scientific | 88-861-043 | Speed used: 20 rpm |
pHrodo™ Red E. coli BioParticles™ Conjugate for Phagocytosis | Life Technologies | P35361 | |
Schneider's Drosophila cell culture media (1x) | Gibco | 21720-024 | Dissection media, combine: Schneiders, FBS, and pen/strep; filter sterilize |
Sodium azide | Sigma-Aldrich | S2002 | 2mM (or 20%) working |
Spring scissors | Fine Science Tools | 15000-00 | |
Sylgard 184 Silicone elastomer | Electron Microscopy Sciences | 24236-10 | Prepare according to provided protocol |
Tween 20 | Sigma | P1379 | For PBS + 0.1% tween |
Vertical Pipette Puller Model 700C | David Kopf Instruments | 812368 | Heater: 55℃ Solenoid: 45 |
Zeiss AxioImager.Z1 fluorescent microscope | Zeiss | Here: Apotome structural interference system with Zeiss Zen imaging software |