Iniezione di mosche Drosophila adulte: un metodo di consegna composta o etichetta

Abstract

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

Questo video descrive come eseguire un’iniezione di mosche adulte vive e include un protocollo di esempio in cui particelle etichettate e blu trypan vengono iniettate negli addome di mosca per saggiare la fagocitosi.

Protocol

Questo protocollo è un estratto da Nazario-Toole e Wu, Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay, J. Vis. Exp. 1. Preparare le particelle di fluoresceina per l’iniezione Ricostituire 10 mg di particelle batteriche uccise dal calore disponibili in commercio etichettate con fluoresceina (vedi Tabella dei materiali)ad una concentrazione di 10 mg/mL aggiungendo 990 μL…

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.