Journal: bioRxiv

Article Title: Targeting extracellular vesicle delivery to the lungs by microgel encapsulation

doi: 10.1101/2022.09.09.507125

Figure Lengend Snippet: HUVEC and macrophages were exposed to DiR labelled MSC-EVs in vitro . EV uptake by HUVEC was characterized after 24h by (A) flow cytometry and (B) AMNIS analysis revealing the internalization of EVs by a limited population of HUVEC, as quantified (C). Unstimulated macrophages (MØ), IL4 stimulated and LPS/IFNg stimulated macrophages readily took up macrophages as shown in representative flow plots (D) and quantified (E). Data represents mean ± SEM.

Article Snippet: Macrophages were characterized for presence and absence of canonical markers using flow cytometry PE-Cy5 mouse anti-rat CD45 (BD Biosciences, Mississauga, ON, Canada), BV786 mouse anti-rat CD11b/c (BD Biosciences, Mississauga, ON, Canada), and PE mouse anti-rat CD34 (Novus Biologicals, Littleton, CO, USA). ( Supplemental Figure 2 ).

Techniques: In Vitro, Flow Cytometry

Journal: bioRxiv

Article Title: Targeting extracellular vesicle delivery to the lungs by microgel encapsulation

doi: 10.1101/2022.09.09.507125

Figure Lengend Snippet: Lungs were isolated, digested, and prepared for flow cytometry analysis 24h after DiR labelled EV administration. (A) Representative flow plots demonstrate consistent proportions of lung endothelial (CD45-CD31+), immune (CD45+CD31- or CD31lo), and other (CD45-CD31-) populations, which was quantified as cell counts, and proportions (B) demonstrating increased numbers of immune cells with the presence of microgels. EV uptake was assessed by evaluation of DiR labelling, representative flow plots are shown (C) and quantification (D) demonstrates significant EV uptake by CD45+CD31- and CD45+CD31lo cells when exposed to encapsulated EVs. Data represented as mean ± SEM, * p<0.05 PBS, Free EVs, and Empty Capsules compared to Encap. EVs, *** p<0.0005 all Tx compared to Encap EVs, # p = 0.07 healthy compared to Encap EVs.

Article Snippet: Macrophages were characterized for presence and absence of canonical markers using flow cytometry PE-Cy5 mouse anti-rat CD45 (BD Biosciences, Mississauga, ON, Canada), BV786 mouse anti-rat CD11b/c (BD Biosciences, Mississauga, ON, Canada), and PE mouse anti-rat CD34 (Novus Biologicals, Littleton, CO, USA). ( Supplemental Figure 2 ).

Techniques: Isolation, Flow Cytometry

Journal: PLoS ONE

Article Title: The novel multi-cytokine inhibitor TO-207 specifically inhibits pro-inflammatory cytokine secretion in monocytes without affecting the killing ability of CAR T cells

doi: 10.1371/journal.pone.0231896

Figure Lengend Snippet: A) Effects of PSL and TO-207 on the cytotoxicity of CAR T cells. K562/CD19/fLucEGFP cells (3 × 10 3 ) were co-cultured with CD4 + or CD8 + CAR T cells (1.5 × 10 4 ), and treated with different concentrations of PSL and TO-207. Viable target cells were quantified using the luciferase assay after 72 h of co-culture. Values were normalized to viable K562/CD19/fLucEGFP cells cultured alone. The error bars represent SDs from three independent experiments. B) Effects of PSL and TO-207 on CAR T cell degranulation. K562/CD19 cells (5 × 10 5 ) and CAR T cells (5 × 10 5 ) were co-cultured in 500 μl T-cell expansion medium in the absence or presence of PSL or TO-207. Following 68 h of co-culture, monensin (2 μM) and APC–anti-human CD107a antibody were added. Cells were incubated for an additional 4 h, and membrane expression of CD107a was determined by flow cytometry. Representative data of four independent experiments are shown. C) Mean fluorescence intensity (MFI) of samples in Fig 5B. The error bars represent SEs from four independent experiments. D) Sustained cytotoxicity of CAR T cells following TO-207 treatment. K562/CD19/fLucEGFP cells (1 × 10 4 ), CAR T cells (5 × 10 4 ), and CD14 + cells (5 × 10 4 ) were co-cultured in the absence or presence of PSL or TO-207 for 72 h, and viable target cells were quantified by the luciferase assay. Values were normalized to the well containing K562/CD19/fLucEGFP cells alone. The error bars represent SDs from three independent experiments. The linear dose-response relationship was assessed using log-transformed dose values (to the base 10) in a mixed model, in which the zero dose was replaced by the log (minimal dose) - 1. P < 0.05 was considered statistically significant. n.s.: not significant.

Article Snippet: Flow cytometry was performed using allophycocyanin (APC)-conjugated IgG 1 (BioLegend), APC–anti-human CD19 antibody (BioLegend), APC–anti human CD107a (BioLegend), APC–Cy7–anti-human CD3 antibody (BioLegend), V500–anti-human CD4 antibody (BD Biosciences, San Jose, CA, USA), and PerCP–Cy5.5–anti-human CD8 antibody (TONBO Biosciences, San Diego, CA, USA).

Techniques: Cell Culture, Luciferase, Co-Culture Assay, Incubation, Membrane, Expressing, Flow Cytometry, Fluorescence, Transformation Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Biotin receptor-mediated intracellular delivery of synthetic polypeptide-protein complexes.

doi: 10.1016/j.jconrel.2023.03.051

Figure Lengend Snippet: Fig. 5. Representative confocal microscopy images of A549 lung epithelial cells incubated with biotin- PEG2k-b-GA30/10 – Cy-5-lysozyme complexes for 1 h, followed by a subsequent application of Alexa 488- conjugated streptavidin (green) in fixed and per meabilized cells (a, b and c). Pearson’s Correlation Coefficient analysis was used for intracellular co- localization studies of labelled protein (Cy5-lyso zyme, red) and AlexaFluor 488 (green) conjugated streptavidin; nuclear stain (DAPI, blue). (For inter pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To monitor cellular internalization of the protein cargo, lysozyme used in this H. Li et al. Journal of Controlled Release 357 (2023) 333–341 experiment was fluorescently labelled using sulfo-Cyanine 5 (Cy5) succinimidyl (NHS) ester (Lumiprobe), according to the manufacturer’s protocol (as described in Supporting information, Methods).

Techniques: Confocal Microscopy, Incubation, Staining