Csu x1 spinning disk confocal head

Larvae or embryos collected from gravid hermaphrodite were mounted onto 3% agarose-padded glass slide, closed with a coverslip and sealed with wax. DIC images shown in Figs 1A, 3A, 3B, 3C, 8A, 8C, 8E and S1A were captured using a Nikon Ti Eclipse widefield microscope equipped with DIC 1.40NA oil condenser and a charged-coupled device camera Cool Snap HQ₂ (Photometrics). All other images and movies were acquired using a spinning disk confocal system composed of a Nikon Ti Eclipse microscope with a CSU-X1 spinning disk confocal head (Yokogawa), DPSS-Laser (Roper Scientific) at 491 and 568 nm excitation wavelengths and an Evolve Rapid-Cal electron multiplying charged-coupled device camera (Photometrics). For both microscopes, Metamorph software (Molecular Devices) was used to control acquisition. Projected images were created using Fiji. All imaging was done at 20°C in an environmental chamber encompassing the microscope stage heated by a JCS temperature controller (Shinko Technos Co, Japan) within a microscope room kept at 18°C by a CITEC precision air conditioning unit.

MOLM-13 cells were cultured on a poly-L-lysine (Sigma-Aldrich) coated 96-well plate. Subsequently, cells were incubated with 15 μg/ml of licMABs or mAb directly labeled with Alexa Fluor 488 (Antibody Labeling Kit, Thermo Fisher Scientific), either on ice-cold water for 120 min or at 37°C for 30, 60 or 120 min. Then, cells were fixed and permeabilized in 20 mM PIPES pH 6.8, 4% formaldehyde, 0.2% Triton X-100, 10 mM EGTA, 1 mM MgCl2 at room temperature for 10 min, followed by incubation in blocking solution (3% bovine serum albumin in PBS). Cells were washed three times with 0.05% Tween-20 in PBS and stored in PBS until examination on a fully automated Zeiss inverted microscope (AxioObserver Z1) equipped with a MS-2000 stage (Applied Scientific Instrumentation, Eugene, Orlando, USA), a CSU-X1 spinning disk confocal head (Yokogawa) and a LaserStack Launch with selectable laser lines (Intelligent Imaging Innovations, Denver, CO). Images were acquired using a CoolSnap HQ camera (Roper Scientific, Planegg, Germany), a 63x oil objective (Plan Neofluoar 63x/1.25) and the Slidebook software (version 6.0; Intelligent Imaging Innovations, Denver, CO). Images were processed with Adobe Photoshop CS4 (Adobe Systems, Mountain View, California, USA).

Immunostaining was performed as described in the Supplemental information. Samples were mounted in Dako mounting medium (DAKO) and analysed using an Eclipse Ti microscope (Nikon) equipped with a 100 x objective, a CSU-X1 spinning disk confocal head (Yokogawa), and a Coolsnap HQ2 camera (Roper Scientific Instruments), controlled by the Metamorph 7.7 software. Analysis by epifluorescence microscopy was performed using a DMRIBe microscope (LEICA microsystems) using 380 nm, 470 nm, or 546 nm LED source excitation, equipped with a Cascade 512 camera (Roper Scientific) driven by the Metamorph 7.7 software. Images were analyzed using the Metamorph software. The levels of EspA and EspD secreted at bacterial-cell contact sites were quantified by delimiting an area corresponding to the bacterial microcolony according to DAPI staining. The integrated fluorescence intensity of relative EspA/EspD staining in the corresponding area was measured and expressed as a ratio to that obtained for DAPI staining.