Poly l lysine coated coverslip

Huh7 cells were cultured on poly-L-lysine coated coverslips (Sigma-Aldrich; Merck KGaA). Following differentiation and treatments, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. Cells were then incubated for 60 min at room temperature with blocking solution (5% FBS in Tris-buffered saline) followed by overnight incubation at 4°C with anti-p62 (1:250; cat. no. PA5-20839; Invitrogen; Thermo Fisher Scientific, Inc.) and anti-p53 (1:250; cat. no. 9286; Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies. Following washing with PBS, cells were incubated with fluorescence-labeled secondary antibodies (Alexa Fluor^® 488-conjugated donkey polyclonal anti-rabbit; 1:500; cat. no. A-21206; and Texas Red-X-conjugated goat polyclonal anti-mouse; 1:500; cat. no. T-6390; both Thermo Fisher Scientific, Inc.) for 2 h at room temperature in the dark. In addition, DAPI (1:1,000; cat. no. D9564; Sigma-Aldrich; Merck KGaA) was used to non-specifically stain the nuclei and samples were incubated with 50 µl DAPI for 10 min at room temperature. Immunostaining was visualized under a fluorescence microscope (magnification, ×400).

Briefly, 1.5 × 10⁵ cells were seeded on Poly-L-Lysine coated coverslips (Cat#P4707, Sigma-Aldrich) in 6 well plates (Cat#3516, Corning). Subsequently, cells were treated with vehicle (dimethyl sulfoxide (DMSO)) (Cat#D128-500, ThermoFisher Scientific) or 10 μM pimozide (Cat#P1793, Sigma-Aldrich) for 24 h. Cells were then fixed in 4% paraformaldehyde (Cat#159-SP, Electron Microscopy Sciences) for 15 min at room temperature. Fixed cells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS) (1x) and then permeabilized in 100% methanol for 10 min at −20°C. Following another DPBS (1x) wash, cells were blocked in blocking buffer (DPBS 1x, 5% normal serum, 0.3% Triton X-100) for 60 min at room temperature. Cells were then incubated with a given primary antibody (diluted 1:100 in DPBS 1x, 1% BSA, 0.3% Triton X-100) for 1 h at 37°C. After three DPBS (1x) washes, cells were incubated with an Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (diluted 1:500 in DPBS 1x, 1% BSA, 0.3% Triton X-100) for 1 h at 37°C (Cat#A-21236, ThermoFisher Scientific). Cells were washed three times with DPBS (1x) before being mounted with ProLong Gold Antifade with 4ʹ6-diamidino-2-phenylindole (DAPI) (Cat#P36931, ThermoFisher Scientific). Using the EVOS FL microscope, images were acquired at 40x objective using the Cy5.5 light cube (ThermoFisher Scientific).

Identification of NETs in this study was similar to a previously published protocol.^{10 (link)} Briefly, 2 × 10⁵ BMDNs were seeded onto poly-l-lysine-coated coverslips (Sigma-Aldrich). In experiments without stimulation or with stimulation with 2% serum (extracted from pristane-treated WT or Mfge8^−/− mice for 6 months), the cells were incubated for 2 h. In experiments with 1 μg/ml LPS, 100 nM PMA or 100 ng/ml MIP-2 stimulation, the incubation was for 4 h. NETs were stained with rabbit anti-histone H3 (citrulline R2+R8+R17) antibody and mouse anti-MPO antibody, followed by incubation with Alexa Fluor 647-conjugated mouse IgG and Alexa Fluor 488-conjugated rabbit IgG (Abcam). DNA was stained with DAPI. Images were collected with Olympus BX51 microscope and Qimaging camera, typically at original × 400 magnification.

Formation of stable conjugates between T cells and autologous tumor cells was analyzed by confocal microscopy. Effector and target cells were co-cultured for 30 min at 1:1 E:T ratio, and then plated on poly-(L-lysine)-coated coverslips (Sigma-Aldrich, Saint-Louis, MO). Cells were then fixed, permeabilized as described¹⁶ (link) and stained with mouse anti-phospho-tyrosine (PY20, BD Biosciences) and rabbit anti-CD8 (Thermo Fisher Scientific), followed by anti-mouse AlexaFluor-488 and anti-rabbit AlexaFluor-647 (Thermo Fisher Scientific). Coverslips were mounted and analyzed using a fluorescence microscope (Leica, HR Sp8) with x63 lenses, and polarization of phospho-Tyr to the immune synapse between T cells and target cells was calculated. Stable conjugates were defined by polarization of p-Tyr at the contact zone between effector cells and tumor cells. Cytotoxic activity was evaluated using the conventional 4 h ⁵¹Cr-release assay as described.¹⁶ (link)