μ slide 8 well chamber

After scraping the fungal colonies on the surface of PDA plates, the mixtures of mycelia and spores were shaken with sterile water and then filtered with sterile 3M filter paper to harvest conidia of the WT and BbVRF1 strains. H. armigera cells (IOZCAS-Ha-I) were cultured in μ-slide 8-well chambers (ibidi GmbH, Germany) by adherent culture (6,000 cells per well). Conidia of WT and BbVRF1 strains were inoculated into the cells (100 conidia per well) and incubated for 72 h. Live cell images were captured by an Andor Dragonfly (Andor, United Kingdom).

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors after informed consent and under Declaration of Helsinki Guidelines (Ethics committee numbers LSHTM-5520 and LSHTM-14576) by using Ficoll-paque PLUS (GE Healthcare, Chicago, IL). Thereafter, monocytes were either enriched by adherence or isolated by positive selection, using human CD14 MicroBeads (Miltenyi, Bergisch Gladbach, Germany) and suspended in RPMI-1640 medium supplemented with pyruvate, L-glutamine, non-essential amino acids, and 10% fetal calf serum.
Cells were seeded into Petri dishes (Corning Inc, NY, USA), 12-well plates (Corning), or μ-slide 8 well chambers (Ibidi, Munich, Germany) and incubated overnight at 37°C in a 5% CO₂ atmosphere for adherence. Cells were then used for morphological and functional assays, as indicated.

For live cell imaging, infected cells were resuspended in filtrated HL5-C and seeded in μ-Slide 8 well chambers (Ibidi) shortly before imaging. Microscopic analysis was performed at 21°C using a Zeiss LSM5 Live confocal microscope with a 100x Europlan apochromat oil immersion objective (N.A. 1.4) and a Diode-Laser 488, as well as a DPSS-Laser 561 [single track mode, 1 Airy unit, dual-band filter (500–545 band pass, 575 long pass)]. Image brightness and contrast were adjusted with ImageJ (Schneider et al., 2012 (link)) to whole images. A minimum of 100 bacteria were quantified at each timepoint except for F.n.n. ΔiglC at 2 hpi (50 bacteria). Imaging of PFA-fixed samples was performed with an Olympus IX81 confocal microscope equipped with an Olympus 100x UPlanSApo oil immersion objective (N.A. 1.4).

Cells were seeded in μ-slide 8-well chambers (Ibidi) and left overnight. The following day, the medium was removed and replaced with carbon dioxide-independent Leibovitz’s L-15 medium (Gibco) supplemented with 10% (v/v) FBS and 100 units/ml of penicillin and 100 µg/ml of streptomycin (Gibco). After 4 h, the medium was removed and 100 nM SiR-DNA (tebu-bio), diluted in L-15, was added to the cells for 15 min. Cells were then washed with PBS before addition of L-15 containing the appropriate drugs and mounting on the incubator chamber of a Deltavision Elite microscope fitted with a 40x/1.30NA U Plan FLN oil objective lens, and operated using SoftWoRx software. Images were captured using a CoolSNAP HQ2 camera (Photometrics) (250 time points, 4 min intervals, 4 × 4 binning, 256 × 256 image size, 4 optical sections (5 μm optical spacing).

We cultured PAEC cells in μ-Slide 8-well chambers (ibidi, Gräfelfing, Germany) and performed the KD experiments by adjusting the volumes. After KD, cells were washed three times in PBS before being fixed with 4% formalin for 10 min at 37°C. After washing the cells six times in PBS, we proceeded to permeabilize them in PBS + BSA 1% (w/v) containing 0.1% Triton X (v/v). Then, we blocked them in PBS + BSA 2% (blocking buffer) for 1 h at room temperature (RT). We incubated the cells with the primary antibodies overnight in blocking buffer, washed three times with blocking buffer for 5 min, and incubated with the secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml) in blocking buffer for 1 h in the dark. Finally, we washed the chambers three times for 5 min in PBS and mounted them in ProLong Diamond Antifade Mountant (ThermoFisher, Waltham, USA). Images were acquired using a Leica DMI6000 inverted microscope with an integrated confocal module SP5 (Leica Microsystems, Germany). The settings used for confocal imaging were maintained in the samples
The following antibodies and dilutions were used are as follows: anti-ET-1 (Abcam, Cambridge, UK, #ab2786, 1:500), phalloidin-Alexa488 (Abcam, #ab22744, 1:1,000), and Alexa Fluor 594-conjugated goat anti-mouse (ThermoFisher, Waltham, USA, #A-11005, 1:1,000).

Cells were seeded in μ-slide 8 well chambers (Ibidi GmbH, Germany) and incubated overnight. Next day, cells were subjected to irradiation (4Gy). Irradiated and control cells (0Gy) were recovered for 3hrs, then fixed with 4% PFA and permeabilized with 0.3% Triton X-100.
Duolink^® Proximity Ligation Assay (Sigma) was carried out using antibodies against γH2Ax and RAD51(Cell Signaling) according to the manufacturer’s instruction. Signals were detected by uorescent microscopy (Nikon Ti2-e Live Cell Imaging System). Quantification of fluorescent signals were carried out by using the Fiji-ImageJ software.