Syto13

Imaging cytometry was performed on a FlowSight (Luminex, Austin, TX, USA) and data were analyzed using IDEAS 6.3 including a machine-learning module (Luminex).
For immune synapse staining HD-derived, isolated T-cells were pre-stimulated with anti-CD2/CD3/CD28-coated beads (Miltenyi Biotec) in the absence/presence of 30 µg/mL CLL-EVs for 48 h at 37 °C, 5% CO₂. Raji cells were labeled with CFSE (ThermoFisher Scientific) and loaded with 1 µg/mL SEA and SEB (both Sigma-Aldrich). T-cells and Raji cells were mixed at a ratio of 1:1, CLL-EVs were added at 60 µg/mL, and samples were incubated for 7 min at 37 °C. Samples were fixed with 1.5% PFA and permeabilized with PBS/2%FCS/0.1% Triton-X. They were stained with surface antibodies and Biotin-XX-Phalloidin (ThermoFisher Scientific), as well as Streptavidin-PE/TexasRed (ThermoFisher Scientific). Staining of nuclei was done using either 7-AAD (Biolegend) or Syto13 (ThermoFisher Scientific). Synapse staining of CAR-T-cells was similarly performed using Mec-1 cells as the target instead of Raji with a CD19 surface staining for detection.

Anti-eNOS (ab5589) and anti-iNOS (ab3523) used for immunohistochemistry were from Abcam (Paris, France). Anti-eNOS antibody (AF950) used for immunoprecipitation experiments was from R&D Systems (Bio-Techne, France). Anti-glutathione antibody recognizing GS-S-proteins was from Virogen (Watertown, MA, USA). Secondary antibodies anti-mouse and anti-rabbit HRP-conjugated were from Cell Signaling Technology (Ozyme, France). Anti-Von Willebrand Factor (VWF) (AB7356) was from Chemicon (Merck Millipore) and anti-VEGF was from Sigma. Secondary anti-goat HRP-conjugated was purchased from Southern Biotech (Clinisciences, France). Secondary Alexa Fluor antibodies (488 and 546) were from Life Technologies (Courtaboeuf, France). Dihydroethidine (DHE), DAF-FM diacetate (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate), dithiotreitol (DTT), 4,6-Diamidino 2-phenylindole dihydrochloride (DAPI), oxypurinol, VAS2870, L-NAME (N-Nitro-L-arginine methyl ester hydrochloride), BH4 (tetrahydrobiopterin dihydrochloride) were from Sigma-Aldrich (Saint Quentin Fallavier, France). 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA) and SYTO-13 were from Thermofisher (Villebon sur Yvette, France), NOC-18 (diethylenetriamine/nitric oxide adduct; DETA NONOate), was from Santa Cruz Biotechnology (Clinisciences, France).

For GeoMx DSP slide preparation, we followed the GeoMx DSP slide prep user manual (MAN-10087-04). Briefly, tissue slides were baked in a drying oven at 60 °C for 1 hour and then loaded to Leica Biosystems BOND RX FFPE for deparaffinization and rehydration. After the target retrieval step, tissues were treated with Proteinase K solution to expose RNA targets followed by fixation with 10% NBF. After all tissue pre-treatments were done, tissue slides were unloaded from the Leica Biosystems BOND RX and incubated with RNA probe mix (COVID-19 Immune Response Atlas panel) overnight. The next day, tissues were washed and stained with tissue visualization markers; CD68-647 at 1:400 (Novus Bio, NBP2-34736AF647), CD45-594 at 1:10 (NanoString Technologies), PanCK-532 at 1:20 (NanoString Technologies) and/or SYTO 13 at 1:10 (Thermo Scientific S7575).

Changes in intracellular calcium concentration were measured in ES cells plated in SL on fibronectin-coated membranes for 24 h. After 30 min staining using 10 nM X-Rhod-1 (Thermo Fisher), the media was replaced with SL with 10 mM HEPES (Sigma) at pH 7.4 containing 2 µM Hoechst 33342. 30 min later, cells were imaged on a Leica CTR7000 HS epifluorescence microscope using a 20×/0.4 n.a. objective. Images were first taken before stretching. Then the membrane was stretched, and the field of view and the focal plane were adjusted. This lasted approximately 1 min, and then images were taken of cells in a stretched state. Images were analysed using Volocity (PerkinElmer) and Fiji [27 (link)], with the X-Rhod-1 signal used to identify cells' calcium signal. To change the intracellular calcium concentration, cells were treated for 30 min with 30 µM BAPTA/AM (Insight Biotechnology). To measure nuclear shape and alignment, cells were incubated with Syto13 (ThermoFisher), at a 5 µM concentration for 30 min, then imaged immediately (control) or stretched and imaged 90 min later (stretched). An ellipse was fitted to the nucleus (Fiji) and the major and minor axes were extracted. To measure the degree of apoptosis during stretching, CellEvent caspase-3/7 green dye (R37111, Thermofisher) was added to the medium 30–60 min prior to stretching.