Cytochalasin d

The survival of NETs-entrapped bacteria was examined as reported previously (17 (link)). Briefly, bacterial cells were cultured in LB medium to an OD₆₀₀ of 0.8 and harvested by centrifugation. The bacterial cells were washed with PBS for three times and resuspended in PBS. Neutrophils (2 × 10⁵ cells/well in a 200 μl volume) were seeded in 96-well cell culture plates and allowed to adhere for 60 min at 22°C. The cells were then stimulated with 1 μg ml⁻¹ PMA for 2 h and centrifuged at 400 g for 5 min, and 150 μl of supernatant was discarded. Then, 50 μl L15 medium containing or not containing 100 U ml⁻¹ DNase I was added to the cells. The cells were maintained at 22°C for 20 min, then cytochalasin D (20 μg ml⁻¹) (Invitrogen, Carlsbad, CA, USA) and 2,000 CFU bacteria (P. fluorescens, V. harveyi, or E. tarda) were added to the plates. The plates were centrifuged at 800 g for 10 min to allow intimate contact of the bacteria with NETs/neutrophils. The plates were then incubated at 22°C for 2, 4, 6, and 8 h. After incubation, the content of each well (bacteria plus neutrophils and NETs) was taken out and serially diluted, and the dilutions were plated on LB agar plates. The plates were incubated at 28°C for 24 h, and the colonies that emerged on the plates were counted. The genetic nature of the colonies was verified by PCR and sequence analysis of the PCR products.

Day 8 iTF-Microglia were used for all phagocytosis assays. iTF-Microglia medium was prepared with pHrodo-Red-labeled synaptosomes at a concentration of 1 mg ml⁻¹ or 0.5 μl ml⁻¹ media of Fluoresbrite Carboxylate YG 1.0 Micron Microspheres (15702-10; Cat. No. 15702-10). After replacing the media with the substrate media, iTF-Microglia were incubated for 1.5 h in the incubator if not otherwise stated. Cells were washed twice with DPBS, dissociated, resuspended in ice-cold DPBS and analyzed via flow cytometry. Where indicated, actin polymerization was inhibited by pretreating cells with 5 μM Cytochalasin D (Invitrogen; Cat. No. PHZ1063) for 30 min before the addition of phagocytic substrate media. For analyzing phagocytic capabilities within microglia clusters, pHrodo-Red-labeled synaptosomes at a concentration of 1 mg ml⁻¹ were added to iTF-Microglia for 1.5 h. Microglia were washed three times with PBS before incubating them in iTF-Microglia media supplemented with 1:2,000 GolgiPlug (BD; Cat. No. 555029) for 4 h. Cells were dissociated, fixed and stained for CCL13 and SPP1 as described above. Flow cytometry data were analyzed using FlowJo, raw median fluorescence intensity values of phagocytosing cells were normalized to no-substrate control samples and data were plotted as fold change using Prism 8.

To determine phagocytic activity, 50′000 BV-2 cells/well were seeded (100 µL per well) in a 96 well plate (Nunclon Micro well 96-well, 167008, Thermo Scientific) and incubated in 5% FBS medium (RPMI-1640, 21875–034, Gibco) with 0.1 µM, 1 µM or 10 µM Lyso-Gb3 or Gb3 and/or 10 µM Cytochalasin D (PHZ1063, Invitrogen) over night at 37 °C (humidified atmosphere with 95% air and 5% CO₂). pHrodo BioParticles (100 µL of 0.5 mg/mL, A10010, Invitrogen) were added to each well and incubated for 2 h at 37 °C. pHrodo uptake was measured using an Infinite M200 Pro microplate reader (TECAN) using 560 nm and 585 nm as excitation and emission wavelengths, respectively. Multiple reads per well were acquired in bottom mode, 16 flashes and 150 gain. Normalized relative fluorescence units (RFU) were obtained by subtracting the average fluorescence intensity of the no-cell negative control and normalizing against the added-Cytochalasin D control per condition.

Bacteria [E. coli, Edwardsiella tarda (Ed. tarda) and S. aureus] were cultured in LB medium until OD₆₀₀ was 0.5 (1 × 10⁹ CFUs mL⁻¹) and enriched in L15 medium. Leukocytes (2 × 10⁵ cells well⁻¹) from the head kidney were distributed in 96-well cell culture plates (Corning) and allowed to settle for 60 min at room temperature. The leukocytes were treated with 500 ng mL⁻¹ PMA for 2 h to induce extracellular trap release, and the ET-negative group was not stimulated with PMA. Subsequently, 20 μg mL⁻¹ of cytochalasin D (Invitrogen, Carlsbad, CA, USA) and anti-fBD (1:1000) were added to the wells. After incubation for 2 h, 2000 CFUs of bacteria in 100 μL of L15 medium were added to the corresponding wells. After incubation for 2, 4 or 8 h, the solution from each well was diluted and spread on bacterial culture plates. After incubation for 12 h, the colonies that appeared on the plates were then recorded according to a reported previously method [32 (link)].

The cell viability and proliferation were detected by MTT colorimetric assay. Briefly, the TIGKs or the hGFBs with cell density of 50,000 cells/well were treated with different concentrations of genipin (G4796, Sigma-Aldrich) (10, 20, 40, 80, 100, and 200 µM) or cytochalasin D (C2168, Sigma-Aldrich) (50, 100, 200, 400, and 800 nM) in CnT-07 medium or fibroblast medium, and cultured in 12-well plate (150,628, Thermofisher Scientific) for 48 h. By the end of culture, the cells were washed with PBS, subsequently incubated with 2 mL of 0.5 mg/mL MTT reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (M2128, Sigma-Aldrich) in 37 °C incubator for 3 h. Then the MTT formazan salt was dissolved in 1 mL of dimethylsulphoxide/ethanol (1:1, v/v), and the absorbances were measured at 595 nm with a 96-well plate reader (82.1581, Sarstedt, Newton, NC, USA). Three replicates for every sample were used in the measurement, and the measurement was repeated three times from three different cell experiments.
The size of the TIGKs and the hGFBs were measured with Countess automated cell counter (Invitrogen, Waltham, MA, USA) after cultivation at different concentrations of genipin or cytochalasin D for 48 h in the 2D monolayer culture. The measurements were repeated three times from three different cell cultures.