Nanoglo live cell buffer

HiBiT-tagged effector proteins were overexpressed in L. pneumophila using the plasmid pxDC61. L. pneumophila strains were grown for 2-3 days on BCYE-Agar plates supplemented with “BCYE Growth Supplements” (Oxoid) and appropriate antibiotics. Gene expression was induced by incubation of bacteria at 37 °C on BCYE agar plates including 0.5 mM IPTG for 24 hours. 0.5 OD units of L. pneumophila were suspended in 500 μl PBS containing 10 μg/ml DNase I, 1 mM MgCl₂, and 1:100 protease inhibitor cocktail. To obtain total luminescence 2 mg/ml lysozyme and 0.5 % (v/v) Triton-X 100 was added to the appropriate samples. All samples were incubated for 30 min at 4 °C. The luminescence was measured in a 384 well plate using NanoGlo® live cell buffer (Promega) supplemented with 1:50 of either a membrane impermeable substrate (Promega), for the periplasmic samples, or the lytic substrate (HiBiT lytic working solution, Promega), for the total luminescence samples.

HiBiT-tagged effector proteins were overexpressed in L. pneumophila using the plasmid pxDC61. One day prior to infection, RAW 264.7 macrophages expressing LgBiT [28 (link),29 (link)] were seeded in a white clear-bottom 96 well cell culture plate (Greiner) at 8 x 10⁴ cells/well. L. pneumophila strains were grown for 2-3 days on BCYE-Agar plates supplemented with “BCYE Growth Supplements” (Oxoid) and appropriate antibiotics. Gene expression was induced by incubation of bacteria at 37 °C on BCYE agar plates with 0.5 mM IPTG for 24 hours. 100 μl of bacteria resuspended in HBSS + DrkBiT (1:1000) at 1.6x10⁸ cfu/ml were used to infect the RAW 264.7 macrophages (MOI = 200). After centrifugation at 300 x g for 8 minutes, 25 μl of NanoGlo® live cell buffer (Promega) supplemented with 1:20 of the extended live cell substrate Endurazine^™ (Promega) was added to each well. Luminescence was measured at 37°C and 5 % CO₂ every 15 minutes for 12 hours in a Tecan Spark plate reader.