[an error occurred while processing this directive] Drosophila Immune Response Data

Materials and Methods for Septic Injury


Materials and Methods for Septic Injury

Septic injury

Septic injury triggers a rapid and widespread response in Drosophila adults. In addition to the introduction of microbes inside the body cavity, this stimulus results in an injury and in the activation of melanisation reactions at the wound site. Consequently the response to septic injury might involve, together with defense genes required to combat infection, genes activated by physical injury. We currently use a mixture of Gram negative (E. coli) and Gram positive (Microccocus luteus) bacteria to extensive immune response.

In this study septic injury was performed by pricking the thorax Drosophila OregonR adult males, aged 3-4 days at 25°C, with a needle previously dipped into a concentrated bacterial culture of Escherichia coli and Micrococcus luteus. Flies were then incubated at 25°C and collected at specific times after infection (1.5, 3, 6, 12, and 48 hours).

Detailed protocol: To inject adult flies, we use a thin metal need le, mounted on a small handle. The bacterial pellet is deposited inside a cut-off microfuge tube lid. We dip the thin needle into the bacterial pellet and puncture the dorsal or lateral side of the thorax of a CO2-anesthetized fly, then gently separate the fly from the needle with a paint brush (See figures). The use of cold light is recommended to avoid dessication of the challenged animals. Flies are then transferred to a clean vial of standard corn-meal fly medium where they normally recover within 5 to 20 minutes. Less than 5% of them may immediately die when injected with a non-pathogenic strain probably due to gross inner lesions. Flies that recover exhibit normal viability. The site of injury heals and remains visible as a small dark spot. This method is straightforward and allows large samples to be tested in a short period of time, i.e. 300 flies or more can be infected per hour.

References

Lemaitre, B., Reichhart, J., and Hoffmann, J. (1997). Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms., Proc Natl Acad Sci USA 94, 14614-14619.

Tzou P., Meister M., Lemaitre B. Methods for studying infection and immunity in Drosophila. Methods in Microbiology in press.