Elyra 7

To carry out SIM imaging, cells were fixed and immunostained as described above. SIM images were captured with Zeiss Elyra 7 (Lattice SIM technology) using either Plan Apo 40×/1.40 oil or Plan Apo 63×/1.40 oil objective and sCMOS camera (PCO Edge). All the single-plane 2D images were captured using Lattice SIM acquisition mode in which 15 phase images were acquired with 1,280 × 1,280 pixel resolution and SIM processing done by ZEN Black (ZEISS) software to give the final super-resolved image. All the representative SIM images presented in the figures were adjusted for brightness and contrast using Adobe Photoshop CS6 software. For live-cell imaging experiments in SIM mode, cells were seeded on a glass-bottom live-cell imaging dish and transfected with indicated plasmids for 12 h. Following incubation, time-lapse imaging was performed in phenol red-free DMEM in a live-cell imaging chamber maintained at 37°C with 5% CO₂ and 95% humidity using Plan Apo 40×/1.40 oil objective.

To prepare the fixed INS-1E β-cell samples, cells were grown on a glass coverslip (ibidi), transfected with plasmids to label ISGs and actin filaments, and stimulated with glucose to acquire three different conditions (basal conditions, the first phase and second phase of GSIS) as described above. Then the samples were washed with 1xPBS three times at room temperature, and incubated with 4% paraformaldehyde for 20 min. Sequentially the cells were immediately washed three times with 1×PBS and incubated in 2 mL of Hoechst 33342 working buffer (1 μg/mL, Cell Signaling) for 5 min. After that, samples were washed three times with 1xPBS, and the glass coverslip was applied with one drop of ProLong^™ Glass Antifade Mountant (Thermo Fisher Scientific) and transferred onto the glass slide. Finally, the samples were kept at room temperature for 48 hours and later stored at −20°C in the dark. The super-resolution fluorescence images were collected in SIM (structured illumination microscopy) mode on Zeiss Elyra 7 with Lattice SIM, equipped with a PCO edge 4.2 sCMOS camera using a Plan-Apochromat 63x/1.4 Oil DIC M27. Fluorescence channels were Hoechst 33342 (Ex 345–355 nm, Em 450–460 nm), EGFP (Ex 483–493 nm, Em 502–512 nm) and mCherry (Ex 582–592 nm, Em 605–615 nm). Finally, the resolution of the SIM data is x-31.3 nm, y-31.3 nm, and z-90.9 nm.

In order to determine the CW components, triple staining of C. albicans cells was performed. The mannans were stained using ConA dissolved in PBS containing 3% BSA, 1mM Ca²⁺, and Mn²⁺ and streptavidin conjugated with Alexa Fluor-568. Total chitin was visualized using CFW. The unmasked β-glucans were stained according to the protocol of Wagener et al. [42 (link)]. The C. albicans 24 h cultures were centrifuged and washed twice with PBS (4000× g, 5 min) and adjusted to A600 = 1 in PBS with 3% BSA. First, cells were incubated using 5 μg/mL of ConA for 1 h, 37 °C. The cells were washed twice with PBS and treated with 1:200 Alexa Fluor 568 conjugated streptavidin for 1 h, 37 °C. The cells were washed twice with PBS and 5 μg/mL Fc–hDectin-1 was added. After 1 h incubation, the cells were washed twice (4000× g, 5 min) and resuspended in PBS. The Fc-hDectin-1 treated cells were incubated with 1:250 Alexa Fluor 448-conjugated anti-human IgG Fc antibodies for 1 h, 4 °C. After that, the cells were washed twice as above, resuspended in PBS, and stained with CFW (0.025 mM) for 5 min, RT [43 (link)]. The cells were washed twice as above, resuspended in PBS, and concentrated. The observation of the preparations (at least 50 cells for each investigated strain) was performed using a super-resolution microscope (ZEISS Elyra 7 with Lattice SIM; Oberkochen, Germany).

For immunofluorescence assay, A549 or HeLa cells were grown on glass coverslips and infected with SPN or STm strains at MOI ~25 for 1 hour followed by antibiotic treatment for 2 hours. At desired time points after infection (9 hours for SPN infection in A549s and 3 hours for infection with STm in HeLa), cells were washed with DMEM and fixed with ice-chilled methanol at −20°C for 10 min. Further, the coverslips were blocked with 3% bovine serum albumin (BSA) in PBS for 2 hours at room temperature (RT). Cells were then treated with an appropriate primary antibody in 1% BSA in PBS overnight at 4°C, washed with PBS, and incubated with suitable secondary antibody in 1% BSA in PBS for 1 hour at RT. Last, coverslips were washed with PBS and mounted on glass slides along with VECTASHIELD with or without 4′,6-diamidino-2-phenylindole (Vector Laboratories) for visualization using a laser scanning confocal microscope (LSM 780, Carl Zeiss) under 40× or 63× oil objectives. The images were acquired after optical sectioning and then processed using ZEN lite software (version 5.0). Superresolution microscopy was performed similarly using Elyra 7 (Carl Zeiss) in SIM mode. For colocalization analysis, bacteria were scored by visual counting of n > 100 bacteria per replicate.

For live-cell imaging, cells were grown in MatTek dishes and imaged in an Elyra 7 with Lattice SIM2 microscope (Zeiss) equipped with an environmental chamber (temperature controlled at 37°C, humidified 5% CO₂ atmosphere), two PCO.edge sCMOS version 4.2 (CL HS) cameras (PCO), solid-state diode continuous-wave lasers, and a Zeiss Plan- Apochromat 63×/1.4 Oil DIC M27, all under the control of ZEN black software (Zeiss). Biotin (400 μM), Janelia Fluor 646 (200 nM), and/or Alexa Fluor 647-conjugated human transferrin (25 μg/ml; Invitrogen) were added to the cells for some experiments.

To analyze the fungal membrane potential, protoplasts were first prepared. The conidial suspension of the indicated strains in CY (15 mL, 5 × 10⁷ conidia mL⁻¹) was shaken for 20 h at 25 °C and the germinating mycelia were incubated in enzymatic digestion buffer (0.8 M MgSO₄, 1 % w/v Lysing Enzymes from Trichoderma (L1412, Sigma), 0.1 % w/v Snailase (S8280, Solarbio, Beijing, China)) for 2 h with gentle shaking (100 rpm) in dark. Protoplasts were collected by centrifugation for 5 min (1500× g) and then shifted to pH 5 or pH 8 conditions and continually shaken (100 rpm) for another 1 h. 2 mM bis (1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3), Invitrogen, Carlsbad, CA, USA) was added to the sample and incubated for 10 min at 4 °C in the dark. Fluorescence was examined using a confocal laser scanning microscope with 488 nm excitation and 509 nm emission using a confocal Zeiss 980 laser scanning microscope with Elyra7 (Zeiss, Oberkochen, Germany) [28 (link),29 (link)]. Images and fluorescence measurements of the confocal data were captured with ZEISS ZEN 3.2 (blue edition) software (Zeiss, Oberkochen, Germany) under the same parameters.