Mct 150 c

Four weeks old and six weeks old plants were spray inoculated with individual strains (Table 1) or PBS (mock control) for the time course and bacterial load experiment, respectively. For the time course experiment, aboveground plant parts were harvested after 1, 3, 6, 9, 12, 24, 48, and 96 hpi. For the bacterial load experiment, aboveground plant parts were harvested 96 hpi. The plant material was collected in RNase‐free microcentrifuge tubes (MCT‐150‐C, Axygen, Corning, USA) and was then immediately flash frozen in liquid N₂. Two plants from different growth boxes were pooled per tube to form a biological replicate. Three biological replicates were sampled per treatment and time point. Flash‐frozen samples were ground to a fine powder in the collection tube using Teflon pestles (General Lab Supply, Lab Supply, Dunedin, New Zealand). RNA extraction was performed using the Isolate II RNA Plant kit (Bioline, London, England).

dHL-60 cells were cultured in RPMI1640 containing 1% BSA, 1% antibiotics, and 10 mM HEPES (HO887, Sigma-Aldrich). To stimulate NET formation in dHL-60 cells, 100, 200, and 400 nM phorbol myristate acetate (PMA; 601010, Cayman, Ann Arbor, MI) or serum from HD patients was used. As a control, serum from HVs was used. HL-60 cells differentiated for 6 days by ATRA were seeded at 5 × 10⁵ cells in a 24-well plate and incubated for 17 h. Next, cells were treated with 150 U/mL S7 nuclease (601010, Cayman) for 1 h and the supernatant was transferred to a microtube (MCT-150-C, AXYGEN, Corning, NY). Nuclease was inactivated by adding 10 mM EDTA to the supernatant and centrifugation at 500g for 5 min. The supernatant was transferred to a new microtube and stored at – 20 °C until analysis.

The CSF sample collection and assay followed standard protocols and have been described previously.^{49 (link)} In short, after monkeys fasted for at least 8 h, a lumbar puncture was performed at the intervertebral space L3-L4 or L4-L5 using a standard needle. CSF was collected into a 1.5 ml sterile microtube (Axygen, MCT-150-C) and immediately frozen at −80 °C. Blood samples were obtained on the same day as the lumbar puncture in fasting conditions. Whole blood was drawn with a 20 G needle gauge into a 5 ml EDTA tube. Tubes were gently inverted 5–10 times and centrifuged at ×3000 g for 10 min at 4 °C. The supernatant was aliquoted in volumes of 200 μl into EP tubes and immediately frozen at −80 °C. The samples were processed at room temperature. The time between collection and freezing of both CSF and plasma sample was <30 min. All CSF and plasma biomarkers including total tau, p-tau231, NFL, Aβ40, and Aβ42 were analyzed by Simoa platform of G-Bio Co., LTD, Hangzhou, China.

A 5 mL blood sample was collected from the brachial vein of each subject. The subjects were instructed to fast for 8 h before blood samples were collected. Each blood sample in BD Vacutainer^® (SST II Advance Plus Blood Collection Tubes, Becton, Dickinson and Company, Belliver Industrial Estate, Roborough, Plymouth, UK) tube was centrifuged at 2500–3000 rpm for 10 min. The separated plasma was collected into a micro tube (MCT-150-C, Axygen, INC., Union City, CA, USA) and stored in a −80 °C freezer (Ultra Low Temperature Freezer, Operon Co., Ltd., Gimpo, Korea). CK was analyzed using a kit (AceChem CK Kit, YD-Diagnostics Corp., Yongin, Korea) and automated clinical chemistry analyzers (Miura One, I.S.E. S.r.l., Rome, Italy).

All tumour containing tissue blocks were retrieved from the archives at the Department of Clinical Pathology, Vejle Hospital, Denmark, where all specimens originally had been processed, using standardized procedures for diagnostic purposes. In brief, the surgical specimens had been routinely fixed in formaldehyde over night, and 1 through 6 tissue blocks from each tumour had been dehydrated and embedded in paraffin. One 4 μm thick, hematoxylin-eosin (HE) stained tissue slide was cut from each tissue block and reviewed by an experienced pathologist for tumour content (i.e., tumour cell nuclei) in steps of 10%. Tissue blocks with a tumour nuclear fraction, subjectively estimated to be lesser than 30%, were excluded from the study, resulting in the inclusion of 1 through 3 tissue blocks from each surgical specimen.
When cutting the tissue sections, care was taken to avoid contaminating tumour tissue from one case to another. Thus, cleaning of the working area was undertaken after cutting each case. Moreover, the technician changed gloves, replaced the knife on the microtome, and cleaned the microtome after finishing cutting the tissue blocks from individual cases. Tissue sections were placed in microtubes (MCT-150-C; 1.5 ml RNase/DNase/pyrogen safe; Axygen, USA), and transported to Exiqon A/S for further processing.

About 5 × 10⁵ fungal conidia in 5 mL of YPD medium were added in each well of a 6-well plate. Meanwhile, tested compounds (NC, 100 μL of DMSO; Ac, 50 μL of 100 μM arthrocolin solution and 50 μL of DMSO; FLC, 50 μL of 800 μg/mL FLC solution and 50 μL of DMSO; Ac+FLC, 50 μL of 100 μM arthrocolin solution and 50 μL of 800 μg/mL FLC solution) were added in each well and then incubated at 30°C with agitation overnight. Fungal conidia were pelleted at 1,000 × g for 10 min in 15-mL tubes (Corning; catalog no. 430052), and the media was removed. The fungal conidia were transferred to a 1.5-mL tube (Axygen; catalog no. MCT-150-C) and washed with PBS three times. The fungal conidia were used for a series of assays and analysis including cell permeability, apoptosis, transmission electron microscopy, autophagy, mitochondrial membrane potential (MMP), oxygen consumption rate (OCR), reactive oxygen species (ROS), mitochondrial level, ATP, nascent protein detection, protein level, and quantitative real-time PCR (RT-qPCR).

Differentiated cell clusters or islets were digested into single-cell suspension by incubation in TrypLE (Thermo Scientific, 12,563,029) at 37 °C (typically 7–10 min). The TrypLE was quenched with 3–4 volumes of DMEM/F12 (1:1) media and cells were centrifuged for 5 min at 300×g. Before transferred into a 1.5-ml microtube (Axygen, MCT-150-C), cells were washed once in PBS (1 mL). Immunostaining for FACS analysis using anti-SOX17, CXCR4, PDX1, NKX6.1 SOX9, C-peptide, and insulin antibodies was carried out using the Fixation/Permeabilization Solution Kit (BD Biosciences, 554714) from BD according to the manufacturer’s instructions. In short, cells were finally resuspended in 300 μl sorting buffer and filtered into 1.5-ml microtubes, and analyzed by Beckman Coulter flow cytometer (Beckman Coulter, DxFLEX) with at least 20,000 events recorded for each test. Analysis of the results was performed using FlowJo software.