Annexin 5 pe

BMMCs (0.5×10⁶ cells/ml) were placed in HARV-RWVs of an RCCS for SMG or in static culture dishes for 1G and grown in a 37°C–5% CO₂ incubator for 48 h. Thereafter, the cells were stained directly with phycoerythrin (PE)-conjugated anti-mouse c-Kit antibody (BioLegend) or stained at 1h after sensitization with 1 μg/ml IgE (Sigma-Aldrich) followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgE (BioLegend), and then analyzed by flow cytometry. For apoptosis analysis, cells cultured under SMG and 1G for 48 and 168 h were stained with Annexin V (A5)-PE (BioLegend) for 15 min at RT in a buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl₂. The proportions of A5-positive apoptotic cells were analyzed using a BD FACS Calibur system. To trace proliferation, cells were labeled with 5 μM CFSE (Invitrogen), a dye that split equally between the two daughter cells following each cell division, and exposed to SMG, SMG+1G, and 1G for 168 h. For SMG+1G, cells were cultured in HARV-RWV of RCCS, and after 24 h, the vessel was separated from RCCS, placed on the ground under 1G condition, and further incubated for 144 h. After the culture, the cells were stained with annexin V-PE. CFSE dilution in the A5-negative live population was analyzed. All the flow cytometry data were visualized using FlowJo V10 software (Tree Star).

After incubation with ALA alone or ALA plus light, the cells were centrifuged and the supernatants were kept at −80 °C for the isolation, labelling, and measurements of cytokines and exosomes as described in Section 2.7. The cells were then incubated in PBS (2% FBS) with an antibody concentration of 2 µL/mL and AnnexinV concentration of 50 µL/mL. The cells were washed with PBS (2% FBS), centrifuged, and the supernatants removed. The amounts of ALA-induced PpIX (1 h antibody incubation) and cell viabilities (1.5 h antibody incubation) in the individual subsets of PBMCs were measured with 4 antibody/dye combination methods: (1) CD3-FITC (Invitrogen, Carlbad, CA, USA), CD19-PE (ImmunoTools GmbH, Friesoythe, Germany), Fixable viability dye-eF450 (Invitrogen), AnnexinV-AF647 (Life Technologies, Eugene, OR, USA); (2) CD3-FITC, CD56-APC-eF780 (Invitrogen), Fixable viability dye-eF450, AnnexinV-PE (Invitrogen); (3) CD4-FITC (eBioscience, San Diego, CA, USA), CD8-PE (Invitrogen), Fixable viability dye-eF450, AnnexinV-AF647; (4) CD11c-FITC (ImmunoTools), CD14-PE/Cy7 (Invitrogen), Fixable viability dye-eF450, Annexin V-AF647. The measurements were done using a Cytoflex S cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA) with the Cytexpert software (Version 2.1, Beckman Coulter); and the analyses were performed using FlowJo software (Version 10, Treestar, Ashland, OR, USA).

Detection of CellTracker™ Deep Red Dye-stained T cell infiltration was performed via fluorescence microscopy (BZ-X Series, Keyence) at 650 nm (Cy5 channel). For the apoptosis assay, Casp3/7 and Annexin V were utilized. KPC spheroids or PDOs were stained with CellEvent™ Caspase3/7 Detection Reagent (#C10423, Invitrogen) according to the manufacturers’ instructions. In brief, 2 drops of the dye per 1 ml T cell medium was applied and incubated for 1 h at 37 °C before fixation of the matrix patch with PFA. Fluorescent microscopic detection was performed at 530 nm (GFP channel). All images were quantified by automated cell counting of fluorescence positive cells through the z-axis at the level of spheroids/PDOs by using a Fiji Macro code (Supplementary Fig. S8). In average, at least 10 spheroids/PDOs were quantified per condition. For 2D co-culture, at least six different areas within the same culture dish were counted and averaged. Annexin V PE (#88-8102-72, Invitrogen) staining was performed after cell trypsinization of 2D grown KPC cells and used according to the manufacturer’s recommendation.

In total, 1 × 10⁶ DPSCs were labeled with 180 μg/mL of iron oxide and transfected with 1 µL/mL of lipofectamine to determine apoptosis by flow cytometry. In brief, they were trypsinized and transferred into a falcon tube. Using a cold PBS solution, the cells were centrifuged at 200× g for 5 min. The supernatant was removed, and the cell pellet was washed two times with Annexin V-PE early apoptosis detection kit I (BD Biosciences, Billerica, MA, USA) suspended in 0.1 mL of Annexin V binding buffer 1X (Invitrogen, USA). Then, addition of 5 µL of Annexin V-PE (Invitrogen, Carlsbad, CA, USA) and 5 μL of 7-Aminoactinomycin D 7-AAD (Invitrogen, USA) took place. They were slowly vortexed and then left for 15 min at room temperature. In total, 400 μL of Annexin V binding buffer (1×) was added as attachment buffer and was further read by a Fluorescence-Activated Cell Sorting (FACS) Calibur flow cytometer (BD Biosciences, Germany) for an hour, and was finally analyzed by the Flow Jow software. The amounts of early apoptosis were determined as the population percentage of Annexin V+/7-AAD, and late apoptosis as the population percentage of Annexin V+/7-AAD + labeled cells with 180 μg/mL of iron oxide and transfected with 1 µ/mL of lipofectamine.