Stage top incubation chamber

HeLa cells were seeded at 1 × 10⁴ cell/well in a 24-well, glass-bottom Ibidi imaging plate. The following day, HeLa cells were infected with C. trachomatis serovar L2 expressing mScarlet at an MOI of 1 and were transfected with pGP-CMV-GCaMP6m 4 h post-infection. The pGP-CMV-GCaMP6m plasmid was a gift from Douglas Kim and the GENIE project (Addgene plasmid # 40,754; RRID:Addgene_40754)^{54 (link)}. At the desired time post infection, medium was removed from cells, and cells were washed with PBS. Cells were incubated in Ca²⁺-free Ringer’s solution for 15 min. Imaging, solution exchanges, image processing, and fluorescence measurements were performed as described in the Fluo-4, AM Ca²⁺ measurements section. Imaging was performed at 37 °C, 5% CO₂, 92% relative humidity using a stage-top incubation chamber (Okolab).

Data shown in Figs. 1 and 5 were acquired on a Zyla 4.2 sCMOS camera (Andor) with a CSU-W1 spinning disk (Yokogawa) mounted on a Ti2 eclipse microscope (Nikon). The Apo Plan 100 × oil/NA1.4 and 60 × oil/NA1.4 objectives and a 405/488/561/638 nm laser (Omicron) were used in combination with an Okolab stage top incubation chamber (Okolab) in case of live-cell experiments. For SIM (3D structured illumination microscopy) images shown in Figs. 3 and 4, an N-SIM E (Nikon) was used, built on a Ti-Eclipse microscope (Nikon). Data were acquired using a z Piezo drive (Mad city labs), an Apochromat TIRF 100 × Oil/NA 1.49 objective, an Orca flash 4.0 LT sCMOS camera (Hamamatsu), a LU-N3-SIM 488/561/640 laser unit (Nikon) and a motorized N-SIM quad band filter combined with a single 525/50 emission filter using the laser line 488 at maximum output power. Z-stacks were acquired with a step size of 200 nm. Both microscopes were controlled by NIS-Elements software (Nikon). Slice reconstruction (NIS-Elements, Nikon) was performed using reconstruction parameters IMC 0.7, HNS 0.7, OBS 0.2.