Technical Reference #1809

1809.

Rab35 mediates transport of Cdc42 and Rac1 to the plasma membrane during phagocytosis Jaewon Shim; Sun-Min Lee; Myeong Sup Lee; Joonsun Yoon; Hee-Seok Kweon; Young-Joon Kim, Yonsei University, Molecular and Cell Biology, 30(1809), (2010)
Link To Paper

Keywords:
Actin filaments/Drosophila Rab35/Phagocytosis/Rho GTPase.

Materials & Methods:
In vivo phagocytosis assay. In vivo phagocytosis assays were carried out in flies as described previously (18 22). Ten adult male-flies aged 5 days were injected with FITC-conjugated E.coli Bioparticles (1mg/ml 50nl) (Invitrogen) on the ventral side with a sharp capillary using a Picospritzer III injector (Parker Hannifin Cleveland OH). Injected flies were incubated for 1 h at 25°C to allow uptake of the injected fluorescent bioparticles by hemocytes. To quench the fluorescence signal of extracellular bioparticles excess Trypan blue solution (0.4% 200 nl) (Sigma) was injected. Fluorescence was visualized using a Zeiss Axioplan 2 microscope (Carl Zeiss MicroImaging Inc.) fitted with a digital camera (AxioCam) and the Axiovision 4.3 software (Carl Zeiss MicroImaging Inc.). Fluorescence signals around the dorsal vessel were quantified using Image J software (NIH Bethesda MD). The phagocytic index was expressed as the area of the signal corresponding to the sum of the encircled areas.

Microscopic Technique
Electron and Confocal Microscopy

Cell Type(s)
SL2 cells