Journal: Molecules

Article Title: The Proteolytic Activity of Neutrophil-Derived Serine Proteases Bound to the Cell Surface Arming Lung Epithelial Cells for Viral Defense

doi: 10.3390/molecules29184449

Figure Lengend Snippet: MHC I cell surface expression increases after treatment with proteases. A549, H1299, and Jurkat cells were incubated with CatG [10 μg/mL], NE [10 μg/mL], or both for 6 h in PBS, and the MHC I cell surface expression was analyzed using flow cytometry. Data were normalized to isotype control and considered significant at p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***) by using an unpaired one-way ANOVA and Sidak post hoc test. n = 3.

Article Snippet: The following cell lines were used in this study: A549 cells (CCL-185, American Type Culture Collection, ATCC, Manassas, VA, USA), an adenocarcinoma human alveolar basal epithelial cell line; NCI-H1299 cells (H1299, CRL-5803, American Type Culture Collection, ATCC, Manassas, VA, USA), a human epithelial-like, non-small cell lung carcinoma cell line derived from the lymph node; and Jurkat E6-1 cells (Jurkat, human T cell lymphoblasts, ARP-177, BEI Resources; NIAID, NIH, Bethesda, MD, USA [ ]).

Techniques: Expressing, Incubation, Flow Cytometry, Control

Journal: Pharmaceuticals

Article Title: Antibodies Enhance the Suppressive Activity of Extracellular Vesicles in Mouse Delayed-Type Hypersensitivity

doi: 10.3390/ph14080734

Figure Lengend Snippet: OVA-Ts-EVs modulate vesicle-mediated interaction of Raji B cells and Jurkat T cells at the immune synapse. ( A ) CD81-GFP-transfected Raji B cells were pulsed with SEE superantigen for 30 min at 37 °C and then cultured with Jurkat T cells in the presence of OVA-Ts-EVs. Twenty four hours later, cells were stained with viability dye and fluoresceinated antibodies against CD19, and analyzed with flow cytometry ( n = 3). Relative changes in the percentage of CD19 neg GFP pos Jurkat T cells caused by SEE-stimulation of Raji B cells were calculated as follows: (percentage of CD19 neg GFP pos events in SEE-stimulated sample)/(percentage of CD19 neg GFP pos events in unstimulated sample); and shown in the graph. ( B ) CD81-GFP-transfected Raji B cells were left untreated (upper panel) or were treated with OVA-Ts-EVs for 4 h at 37 °C (lower panel), both populations were then pulsed with SEE for 30 min at 37 °C, and mixed with CMAC-stained Jurkat T cells (1 × 10 5 cells) in a ratio 1:1. Then, cell mixtures were plated onto Poly-L-Lys-coated slides for 1 h incubation at 37 °C, fixed, blocked, stained with selected primary and then secondary antibodies, mounted on Prolong Gold and analyzed with confocal microscope; scale bar: 10 µm. ( C ) CD81-GFP-transfected Raji B cells were treated with OVA-Ts-EVs for 4 h at 37 °C, pulsed with SEE for 30 min at 37 °C, and cultured with CMAC-stained Jurkat T cells (1 × 10 5 cells) in a ratio 1:1 for 24 h on standard culture plate. Then, cell mixtures were plated onto Poly-L-Lys-coated slides for 1 h incubation at 37 °C, fixed, blocked, stained with selected primary and then secondary antibodies, mounted on Prolong Gold and analyzed with confocal microscope; scale bar: 10 µm. Data are expressed as mean ± SD. Two-tailed Student t -test; * p < 0.05.

Article Snippet: The human Jurkat T-cell line (E6.1 clone) and the lymphoblastoid Raji B-cell line (Burkitt lymphoma, both acquired from the DSMZ Organization, Braunschweig, Germany, and routinely tested for mycoplasma) were cultured in RPMI 1640 medium supplemented with GlutaMAX, HEPES (Gibco Life Technologies, Grand Island, NY, USA) and 10% fetal bovine serum (FBS, Hyclone, ThermoFisher, Waltham, MA, USA).

Techniques: Transfection, Cell Culture, Staining, Flow Cytometry, Incubation, Microscopy, Two Tailed Test