Celltracker violet bmqc

Samples were fixed in 10% aqueous buffered zinc formalin (Z-Fix, Anatech) for at least 6 h for immunofluorescence staining. Phalloidin conjugates (AF488, ThermoFisher) were used to stain the F-actin of the cell cytoskeleton. For identifying MSC in co-cultures studies, the MSC were stained with CellTracker Green CMFDA (Invitrogen), and fibroblasts were stained with CellTracker Violet BMQC (Invitrogen) before seeding them in microgels. The endothelial sprouts were stained with an endothelial cell-specific marker Ulex Europaeus Agglutinin I (UEA-I, Vector Laboratories). DAPI (Invitrogen) was used as a nuclear counter-stain. Before staining, the fixed samples were washed and permeabilized with 0.1% Triton X-100 (Sigma) in 10 mM PBS. The samples were then stained for 1 h in the dark, and z-stacks were acquired using a Nikon A1R confocal microscope. All the acquired optical sections were processed using Fiji (ImageJ) software.

Peripheral blood mononuclear cells (PBMCs) were purified by ficoll density centrifugation using LSM1077 (Sigma-Aldrich). NK cells were isolated using a negative selection NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated NK cells were cultured in IMDM medium (Gibco^TM) with 10% FCS, 1% P/S, and 10 U/mL IL-2 and rested at 37 °C overnight. NK cells were pretreated with 100 µg/mL EVs or soluble protein for 24 h before killing assays were performed. For killing assays, K562 target cells were stained with 5 µM CellTracker^TM Violet BMQC (Life Technologies) fluorescent dye in serum-free medium at 37 °C for 45 min. Then, FCS was added to the cells at a final concentration of 20%. Cells were resuspended in fresh medium and seeded accordingly in IMDM medium with 10% FCS and 1% P/S in U-shaped, 96-well plates. NK cells were added to the target cells in ratios ranging from 1.25:1 to 10:1. The cells were cocultured for 3 h before they were harvested and centrifuged at 300× g for 5 min. The supernatant was discarded, and the cells were resuspended in 200 µL PBS before they were stained with 100 ng/200 µL 7-AAD (BioLegend). Cell death was measured by flow cytometry using a FACS Canto II cytometer (BD Bioscience) and analyzed by FACS Diva software. The killing of untreated NK cells was subtracted from the treated samples and induced killing was displayed.

Peripheral blood mononuclear cells (PBMCs) were purified by ficoll density centrifugation using LSM1077 (Sigma-Aldrich). NK cells were isolated using a negative selection NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated NK cells were cultured in IMDM medium (Gibco^TM) with 10% FCS, 1% P/S, and 10 U/mL IL-2 and rested at 37 °C overnight. For killing assays, HL60 cells were stained with 5 µM CellTracker^TM Violet BMQC (Life Technologies) fluorescent dye in serum-free medium at 37 °C for 45 min. Then, FCS was added to the cells at a final concentration of 20%. Cells were resuspended in fresh medium and seeded accordingly in IMDM medium with 10% FCS and 1% P/S in U-shaped 96-well plates. NK cells were added to the target cells in ratios ranging from 1.25:1 to 10:1. The cells were co-cultured for 3 h before they were harvested and centrifuged at 300 × g for 5 min. The supernatant was discarded, and the cells were resuspended in 200 µL PBS before they were stained with 1 µl 50 µg/ml PI per sample. Cell death was measured by flow cytometry using a FACS Canto II cytometer (BD Bioscience) and analyzed by FACS Diva software.

For killing assays, target cells were stained with 5 µM CellTracker^TM Violet BMQC (Invitrogen, Carlsbad, CA, USA) fluorescent dye in serum-free medium at 37 °C for 45 min. Then, cells were washed twice in complete medium and seeded accordingly in IMDM medium with 10% FCS and 1% P/S in U-shaped 96-well plates. NK cells were added to the target cells in ratios ranging from 1.25:1 to 10:1. The cells were co-cultured for 3 h before they were harvested and centrifuged at 300 g for 5 min. The supernatant was discarded and the cells were resuspended in 200 µL PBS before they were stained with 100 ng 7-AAD (Biolegend, San Diego CA, USA). Cell death was measured by flow cytometry using a FACS Canto II cytometer (BD Bioscience, Heidelberg, Germany) and analyzed by FACS Diva software and FlowJo (version 10.6.1, (BD Bioscience, Heidelberg, Germany).

Iron(II) chloride tetrahydrate (FeCl₂∙4H₂O, 99.0%, Aldrich), iron(III) chloride hexahydrate (FeCl₃∙6H₂O, 99.0%, Aldrich), sodium hydroxide (NaOH, Daejung), poly(diallyldimethylammonium chloride) (PDADMAC, MW: 100,000–200,000, 20 wt%, in H₂O, Aldrich), tetramethyl orthosilicate (TMOS, 99%, Aldrich), sodium phosphate dibasic (Na₂HPO₄, 99%, Aldrich), sodium dihydrogen phosphate (NaH₂PO₄, 99%, Aldrich), sodium chloride (NaCl, 99%, Jin Chemical), 11-mercaptoundecanoic acid (MUA, 95%, Aldrich), sulfuric acid (H₂SO₄, 95%, Junsei), hydrogen peroxide (H₂O₂, 30–35%, Junsei), hydrochloric acid (HCl, 35%, Junsei), fluorescein diacetate (FDA, Aldrich), Cell Tracker^TM Green CMFDA (Invitrogen), Cell Tracker^TM Violet BMQC (Invitrogen), Cell Tracker^TM Orange CMRA (Invitrogen), yeast-extract-peptone-dextrose broth (YPD broth, Duchefa Biochemistry), yeast-extract-peptone-dextrose agar (YPD agar, Duchefa Biochemistry), absolute ethanol (99.8%, Merck), and acetone (99.8%, Merck) were used as received. Ultrapure water (18.3 MΩ∙cm) from the Human Ultrapure System (Human Corp.) was used. Permanent neodymium magnets (110 mT (15 × 10 × 1 mm), 270 mT (15 × 10 × 4 mm), and 320 mT (D30 × 10 mm), Umagnet) were used.