Injektion af voksne Drosophila Fluer: En metode til sammensatte eller Label Delivery

Abstract

Kilde: Nazario-Toole, A. E., Wu, L. P. Vurdering af Cellular Immune Response af Fruit Fly, Drosophila melanogaster, Ved hjælp af en In Vivo Phagocytosis Assay. J. Vis. Exp. (2019).

Denne video beskriver, hvordan man udfører en injektion af levende voksne fluer og indeholder et eksempel protokol, hvor mærkede partikler og trypan blå injiceres i flyve maver til assay fagocytose.

Protocol

Denne protokol er et uddrag fra Nazario-Toole og Wu, Vurdering af frugtfluens cellulære immunrespons, Drosophila melanogaster, Ved hjælp af en In Vivo Phagocytosis Assay, J. Vis. Exp. (2019). 1. Forbered fluorescerende partikler til injektion 10 mg kommercielt tilgængelige, varmeaflivede bakteriepartikler mærket med fluorescein (se materialetabel)rekonstrueres til en bestandskoncentration på 10 mg/mL ved at tilsætte 990 μL steril 1x …

Materials

5430-10 PicoNozzle Kit	World Precision Instruments	5430-10	Holder for 1.0mm pipette
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)
Needle Pipette Puller	David Kopf Instruments	Model 725
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.
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.