Systemic bacterial infection assay

Bacterial strain used

• E.coli (OD600:100)

Setting up the primary and secondary cultures

• Take out the bacterial glycerol stock from -80^oC
• Dip a sterilized inoculating loop in the stock, streak the culture on a freshly prepared LB-Agar plate, and incubate the plate at 37^oC overnight.
• Once there is visible growth (single colonies) the next day, take out one single colony, inoculate it in the required amount of liquid LB (Luria-Bertani) media (primary culture), and incubate it at 37^oC in a shaking incubator till the time the optical density at 600 nm (OD600) reaches between 0.4–0.6. 
• Once the primary culture reaches OD600 between 0.4–0.6, take out 100-200ml of the culture, re-suspend it in the required amount of liquid LB media, and incubate at 37^oC in a shaking incubator (secondary culture). 

Note: For each set of experiments, the secondary culture was freshly prepared from the primary culture.

Infection experiments

• For larval infections, once the secondary bacterial culture reached OD600 around 0.5, the cultures were concentrated by centrifugation.
• The pellet formed post centrifugation was re-suspended in phosphate-buffered saline (1X PBS) to the appropriate OD600 value (mentioned above).
• For all the analyses, synchronized third instar larval batches were used. The larvae were washed thrice with sterile ddH2O and pricked at the postero-lateral part using a fine insect pin (Austerlitz insect pins)/tungsten pin: Fine Scientific Tools) dipped in bacterial suspension. 

For proper holding of the pin, it was mounted on a pin holder (Fine Scientific tool: Cat. No. 91606-07). 

Note: The larvae should be pricked gently; the pin should penetrate the cuticle with minimal damage to internal tissues/organs. Before dissection, check out the melanization spots to ensure that the larvae have been infected. The spots that appear at the site of pricking/injury correspond to the activation of the melanization cascade. 

• Mock injections were done using 1X PBS dipped pins.
• Once infected, larval batches were transferred to food plates and reared at 25^oC till dissection. 
• All observations were made 4 hours post-infection for the experiments related to this study (https://doi.org/10.7554/eLife.67158).