Nunc lab tek 2 cc2 chamber slide

Human EVT cell line HTR-8/SVneo (reffered to as HTR), an immortalized first trimester trophoblast cell line, was kindly provided by Graham et al, 21 (link) SGHPL-4 cell line (referred to as SGHPL) by Dash 22 (link) and HIPEC-65 (referred to as HIPEC) cell line by Pavan. 23 (link) All cell lines were cultured as instructed. [21] (link)[22] (link)[23] (link) Trophoblastic cell spheroids were prepared according to Foty, 24 with modifications. Cells with 90% confluency were harvested and counted. Cells resuspended in 20 µL (100 cells/µL) of complete medium or medium containing indicated reagents were seeded onto the bottom of a lid of a 60 mm culture dish as hanging droplets. The lid was then put back onto the PBS-filled bottom chamber of the culture dish and incubated for 72 to 96 hours until spheroids are stably formed. Single spheroids were carefully picked with a 100 µL tip and transferred on Nunc Lab-Tek II CC2 chamber slides (Thermo Fisher Scientific) for microscopic evaluation. Small interfering RNAs (siR-NAs) were transiently transfected as reported. 25 (link) Aurora A inhibitor MLN8054 and Erk1/2 inhibitor PD98059 were used as described, 26 (link) and, together with other reagents and siRNAs, are described in supplementary information.

For indirect immunofluorescence staining, cells were seeded on Nunc Lab-Tek II CC2 chamber slides (Thermo Fisher Scientific) and fixed with 4% paraformaldehyde containing 0.2% Triton X-100 for 15 minutes at room temperature as described. 30 (link) Primary antibodies, cell imaging, and signal intensity measurement are detailed in supplementary information.

Neutrophils were isolated from whole blood of UC patients and NC by negative selection using MACSxpress Beads [Miltenyi Biotec] and were seeded on eight-well Nunc Lab-Tek II CC2 Chamber Slides [Thermo Fisher Scientific] [2.5 × 10 5 cells per well] for 30 min in RPMI1640 medium at 37°C and 5% CO 2 . After adhesion, the cells were either left unstimulated or stimulated with 20 ng/ml tumour necrosis factor-α [TNF-α, R&D Systems], 20 ng/ml interleukin-1β [IL-1β, R&D Systems], 25 ng/ml IL-6 [R&D Systems], 100 ng/ ml interferon-γ [IFN-γ, Peprotech], 100 ng/ml lipopolysaccharide [LPS, Sigma-Aldrich], 1 μg/ml cytosine-phosphate-guanine [CpG] oligodeoxynucleotide [Invitrogen] and 50 nM phorbol myristate acetate [PMA, eBioscience] for 3.5 hs. To isolate NETs, UC neutrophils [2 × 10 6 cells per well] were seeded in six-well plates for 30 min in 1 ml RPMI1640 medium and then stimulated with 50 nM PMA. After 3.5 h, cells were washed twice with fresh RPMI1640 medium and the NETs were collected by extensively pipetting with 1 ml RPMI1640 medium and centrifuged the resulting supernatant [20 × g for 5 min].