Exosome uptake was visualized by fluorescent microscopy after 6 h, which revealed Dil-positivity for 33% of the cancer cells, thereby confirming successful uptake of exosomes into cytoplasmic compartments (Figure 2E,F)
Exosome uptake was visualized by fluorescent microscopy after 6 h, which revealed Dil-positivity for 33% of the cancer cells, thereby confirming successful uptake of exosomes into cytoplasmic compartments (Figure 2E,F). Open in a separate window Figure 2 Isolation and characterization of NKExos. potential to treat ER+ breast tumor. Transfected NK92MI cells produced substantial levels of BCL-2 siRNAs, without considerably influencing NK cell viability or effector function and led to loading of siBCL-2 in NKExos. Remarkably, focusing on BCL-2 via siBCL-2 NKExos led to enhanced intrinsic apoptosis in breast tumor cells, without influencing nonmalignant cells. Collectively, our prototypical results for BCL-2 in breast cancer provide proof of concept for any novel strategy to use NKExos as a natural delivery vector for siRNA focusing on of oncogenes. for 10 min and at 16,000 for 30 min at 4 C. Next, exosomes were concentrated using 100K Amicon? Ultra-15 centrifugal filters (Merck Millipore, Co cork, Ireland) and purified using Exo-spin? Exosome Purification kit (Cell Guidance Systems, Cambridge, UK) relating to manufacturers instructions. Purified exosomes were quantified using the CD63 Tirasemtiv (CK-2017357) Trific? Exosome detection kit (Cell Guidance Systems) relating to manufacturers instructions and stored at 80 C until further use. Immunogold staining and Transmission Electron Microscopy (TEM) was performed from the DKFZ Core Facility Unit Electron Microscopy (Heidelberg, Germany). For immunostaining, CD56 (clone: MEM-188, Biolegend), CD63 (clone: H5C6, BD Biosciences), and isotype settings were used followed by a rabbit-anti-mouse antibody conjugated to 5 nm platinum particles. The samples were analyzed using an EM910 (Zeiss, Oberkochen, Germany) transmission electron microscope at 63,000 magnification. Particle size of exosomes was determined by Nanoparticle Tracking Analysis (NTA) using a ZetaView? Instrument (Particle Metrix, Inning am Ammersee, Germany) by Cell Guidance Systems. 2.7. Exosome Uptake Exosome labeling was performed having a 1 M Vybrant? Dil cell-labeling remedy (Invitrogen) followed by washing and ultrafiltration with 100K Amicon? Ultra-15 centrifugal filters as previously explained [37]. Dil-labeled exosomes were then co-cultured with GFP+ MCF-7 cells for 6 h. Nuclear staining was performed using NucBlue? Live Cell Stain ReadyProbes? reagent (Invitrogen) relating to manufacturers protocol. After incubation, exosome transfected cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) and analyzed by fluorescence microscopy. Image acquisition was performed using an Olympus BX63 microscope and a DP80 video camera (Olympus). Exosome uptake was quantified by counting reddish and green fluorescent positive cells using Image J 1.53a software (National Institutes of Health, Bethesda, MD, USA). 2.8. Live Cell Analysis Cells were treated with siScr or siBCL-2 NKExos, followed by addition of Incucyte? Annexin V Red (1:200) or Incucyte? Caspase-3/7 Red Dye (1:400) to the tradition. For analysis of GFP silencing, GFP+ cells were treated with siScr or siGFP NKExos cells. Live cell imaging was performed with the Incucyte? S3 Systems (Sartorius, G?ttingen, Germany) for Tirasemtiv (CK-2017357) 48 h. 2.9. Caspase-9 Activation Cells (1 104 per well) were plated inside a 96-well plate (NunclonTM Delta Surface, Thermo Fisher Scientific, Waltham, MA, USA) and incubated with the siScr, siBCL-2 NKExos or Staurosporin (2.5 m) (Abcam, Cambridge, UK). After 24 h, Caspase 9 activity was measured using the CaspaseGlo? 9 Assay (Promega, Madison, WI, USA) relating to manufacturers instructions. 2.10. Statistical Analysis Statistical analysis was performed with GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Mean ideals with standard deviation (SD) are demonstrated. The 95% confidence level was used, and normality distribution was determined by the ShapiroCWilk test. = 4). (E) Intracellular staining of NK92MI transfectants with anti-BCL-2 antibodies or Tirasemtiv (CK-2017357) isotype control was analyzed using circulation cytometry. (F) BCL-2 manifestation level of NK92MI-siBCL-2 and NK92MI-siScr cells (= 3) is definitely shown as specific fluorescence indices (SFI) levels calculated by using median fluorescence intensity (MFI) of BCL-2/MFI Isotype control. (G) NK92MI transfectants were analyzed for 7-AAD positivity by circulation cytometry. Living cell human population is definitely demonstrated by exclusion of 7-AAD+ deceased cells. (H,I) NK92MI transfectants were incubated with K562 cells at a different effector to target (E:T) ratios. Lysis of K562 cells was determined by 2 h Tirasemtiv (CK-2017357) cytotoxicity assays. (H) TSPAN12 Exemplary results and (I) combined data at an E:T percentage of 2.5:1 are demonstrated. 3.2. Characterization of NK Cell-Derived Exosomes (NKExos) For production of exosomes, NK92MI-siBCL-2 and NK92MI-siScr cells were cultured for 72.