An In Vivo Assay to Quantify Bacterial Phagocytosis in Adult Drosophila Flies

Published: February 29, 2024

Abstract

Source: Nazario-Toole, A. E., et al. Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay. J. Vis. Exp. (2019)

This video demonstrates an in vivo assay designed to measure bacterial phagocytosis in adult Drosophila flies. Following the injection of fluorescently labeled bacteria into the abdomen of adult flies, hemocytes associated with the dorsal vessel engage in phagocytosis. The introduction of trypan blue dye serves to quench the fluorescence emitted by extracellular, non-phagocytosed bacteria. This quenching process enhances the identification of phagocytosed bacteria when observed under a fluorescence microscope.

Protocol

1. Prepare Fluorescein particles for injection Reconstitute 10 mg of commercially available, heat-killed bacteria particles labeled with fluorescein (see Table of Materials) to a stock concentration of 10 mg/mL by adding 990 µL sterile 1x PBS and 10 µL 50 mM sodium azide. Vortex to mix. Divide into single-use 8 µL aliquots in 0.2 mL tubes and store in a dark box at 4 °C to minimize light-associated sensitivity. NOTE: So…

Declarações

The authors have nothing to disclose.

Materials

0.2μm Red Fluorescent Carboxylate Modified FluoSpheres Invitrogen F8810 Fluorescently-labeled latex beads to test general phagocytic capacity of phagocytes. (~580/~605 nm) Inject a 1:20 dilution in PBS with 5% dye.
5430-10 PicoNozzle Kit World Precision Instruments 5430-10 Holder for 1.0mm pipette
E. coli (K-12 Strain) BioParticles, Alexa Fluor 488 conjugate Invitrogen E13231 Killed E. coli labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~495/~519 nm)
E. coli (K-12 Strain) BioParticles, Alexa Fluor 594 conjugate Invitrogen E23370 Killed E. coli labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~590/~617 nm)
E. coli (K-12 Strain) BioParticles, Fluorescein conjugate Invitrogen E2861 Killed E. coli labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~494/~518 nm)
E. coli (K-12 Strain) BioParticles, Texas Red conjugate Invitrogen E2863 Killed E. coli labeled with Texas Red. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~595/~615 nm)
E. coli (K-12 Strain) BioParticles, Texas Red conjugate Invitrogen E2863 Killed E. coli labeled with Texas Red. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~595/~615 nm)
Needle Pipette Puller David Kopf Instruments Model 725
pHrodo Red E. coli BioParticles Conjugate for Phagocytosis Invitrogen P35361 Killed E. coli labeled with pHrodo Red. Use to test phagocyte reconition, uptake, and phagosome maturation of gram-negative bacteria. (~560/~585 nm). No need to quench with Trypan Blue.
pHrodo Red S. aureus BioParticles Conjugate for Phagocytosis Invitrogen A10010 Killed S. aureus labeled with pHrodo Red. Use to test phagocyte reconition, uptake, and phagosome maturation of gram-positve bacteria. (~560/~585 nm). No need to quench with Trypan Blue.
Pneumatic PicoPump PV820 World Precision Instruments SYS-PV820 The World Precision Instruments Pneumatic PicoPump PV820 uses differential pressures to hold liquid in the glass needle between injections. The user manually controls short bursts of gas pressure to expel the liquid – allowing delivery of sub-nanoliter volumes. The amount of liquid delivered depends on two main variables – the size of the glass needle opening and the amount of time injection pressure is applied. set the instrument to 100 ms "TIMED" mode.
S. aureus (Wood Strain without protein A) BioParticles, Alexa Fluor 488 conjugate Invitrogen S23371 Killed S. aureus labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~495/~519 nm)
S. aureus (Wood Strain without protein A) BioParticles, Alexa Fluor 594 conjugate Invitrogen S23372 Killed S. aureus labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~590/~617 nm)
S. aureus (Wood Strain without protein A) BioParticles, Fluorescein conjugate Invitrogen E2851 Killed S. aureus labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~494/~518 nm)
Thin Wall Glass Capillaries World Precision Instruments TW100F-3 Needles for injection. OD = 1.0 mm
Trypan Blue Solution (0.4%) Sigma T8154 Used to quench extracellular fluorescence of Fluorescein, Alexa Fluor, or Texas Red labeled particles.
ZEISS SteREO Microscope (Discovery.V8) Zeiss SteREO Discovery.V8 Inverted fluorescence microscope for imaging flies. Use a digital camera (example: AxioCam HC camera) and the accompanying software (example: AxioVision 4.7 software) to take pictures.
Zymosan A (Saccharomyces cerevisiae) BioParticles, Alexa Fluor 488 conjugate Invitrogen Z23373 Killed labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of yeast. (~495/~519 nm)
Zymosan A (Saccharomyces cerevisiae) BioParticles, Alexa Fluor 594 conjugate Invitrogen Z23374 Killed labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of yeast. (~590/~617 nm)
Zymosan A (Saccharomyces cerevisiae) BioParticles, Fluorescein conjugate Invitrogen Z2841 Killed labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of yeast. (~494/~518 nm)
Zymosan A (Saccharomyces cerevisiae) BioParticles, Texas Red Invitrogen Z2843 Killed labeled with Texas Red. Use to test phagocyte recogntion and uptake of yeast. (~595/~615 nm)

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An In Vivo Assay to Quantify Bacterial Phagocytosis in Adult Drosophila Flies. J. Vis. Exp. (Pending Publication), e21972, doi: (2024).

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