Journal: Biomedicines

Article Title: Morphological and Immunocytochemical Characterization of Paclitaxel-Induced Microcells in Sk-Mel-28 Melanoma Cells

doi: 10.3390/biomedicines12071576

Figure Lengend Snippet: Primary and secondary antibodies.

Article Snippet: Caspase-6 , Primary antibody: rabbit anti-human, rabbit polyclonal , ALEXA FLUOR ® 555 conjugated Ex.553 nm/Em.568 nm , 1:100 , BS-0151R-A555, Bioss antibodies/Nordic BioSite, Täby, Sweden.

Techniques: Concentration Assay, Recombinant

Journal: Cancers

Article Title: The RHOA Mutation G17V Does Not Lead to Increased Migration of Human Malignant T Cells but Is Associated with Matrix Remodelling

doi: 10.3390/cancers15123226

Figure Lengend Snippet: ( A , B ) Volume, track speed, track length, and displacement of T-cell lymphoma cell lines expressing RHOA-G17V in native lymphoid tissue. ( A ) Volume of cell bodies (means). Each dot represents the volume of one cell from three independent experiments. * p = 0.039, Mann–Whitney-test. ( B ) Mean track speed of the empty-vector-transfected or RHOA-G17V-overexpressing HuT78 and HH cells compared with the endogenous human-CD3-positive T cells and the CD19-positive B cells. Each dot represents the mean track-based speed of one cell; the cells were recorded in three independent experiments. ** p < 0.01, Kruskal–Wallis test with Dunn´s post-test for multiple comparisons. ( C ) Track length of the empty-vector-transfected or RHOA-G17V-overexpressing HuT78 and HH cells compared with endogenous human-CD3-positive T cells and the CD19-positive B cells. Each dot represents the track length of one cell; the cells were recorded in three independent experiments. * p < 0.05, *** p < 0.001, Kruskal–Wallis test with Dunn´s post-test for multiple comparisons. ( D ) Displacement of the empty-vector-transfected or RHOA-G17V-overexpressing HuT78 and HH cells compared with the endogenous human-CD3-positive T cells and CD19-positive B cells. Each dot represents the track length of one cell; the cells were recorded in three independent experiments. *** p < 0.001, Kruskal–Wallis test with Dunn´s post-test for multiple comparisons. ( E ) Examples of empty-vector-transfected HuT78 cells (green) with reconstructed tracks of a 15 min movie in native lymphoid tissue. ( F ) Examples of RHOA-G17V-expressing HuT78 cells (pink) with reconstructed tracks of a 15 min movie in native lymphoid tissue.

Article Snippet: Endogenous B and T cells were labelled with Alexa Fluor 555-anti-human CD19 (polyclonal rabbit antibody; Bioss Antibodies, Woburn, MA, USA) and Alexa Fluor 647-anti-human CD3 (clone UCHT1; BD Biosciences, Franklin Lakes, NJ, USA), as previously described.

Techniques: Expressing, MANN-WHITNEY, Plasmid Preparation, Transfection

Journal: Journal of Extracellular Vesicles

Article Title: HIV‐1 Nef is carried on the surface of extracellular vesicles

doi: 10.1002/jev2.12478

Figure Lengend Snippet: Characterisation of Nef EVs. (a) Nef EVs, obtained through ultracentrifugation from Nef‐transfected HEK293T cells, underwent fractionation using iodixanol gradient. 13 fractions were collected, and Nef concentrations (pg/mL) along with particle concentrations (particles/mL) for the first twelve fractions were determined through Nef ELISA and nanoparticle tracking analysis, respectively. (b) Fractionated on the iodixanol gradient Nef EVs were subjected to Western blot analysis for the presence of Alix, CD63, TSG101, Hsp70, Nef, CD9 and CD81. (c) Nef EVs were analysed utilising the ExoView platform. Particle capture was achieved using anti‐CD63, anti‐CD81, or anti‐CD9 antibodies, followed by detection with fluorescently labelled anti‐CD63‐CF647 (red), CD81‐CF555 (green) or CD9‐CF488 (blue). Anti‐IgG served as a capture control. The graph depicts means with standard errors of the means (SEM) for the number of EVs positive for CD63 (red), CD81 (green) and CD9 (blue) from three capture spots in two independent measurements.

Article Snippet: Chips were washed three times and incubated with anti‐CD9‐CF488 (Abcam, cat. # ab267502), anti‐CD81‐CF555 (Bioss, cat. # BS‐6934R‐A555) and anti‐CD63‐CF647 (Abcam, cat. # ab233056) following the manufacturer's instructions.

Techniques: Transfection, Fractionation, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Journal of Extracellular Vesicles

Article Title: HIV‐1 Nef is carried on the surface of extracellular vesicles

doi: 10.1002/jev2.12478

Figure Lengend Snippet: Nef localisation on the EV surface. (a) Nef EVs, obtained from HEK293T cells transfected with the Nef vector, were divided into two equal volumes and treated with proteinase K. The quantification of Nef associated with Nef EVs in the presence or absence of 0.5% Triton X‐100 was performed using ELISA. One representative experiment from two performed is presented. (b) The amount of syntenin associated with Nef EVs in the presence of 0.5% Triton X‐100 was quantified using ELISA. One representative experiment from two is shown. (c) Triplicate samples of EVs produced by Nef‐transfected SupT1 cells were treated or not with proteinase K, and analysed by Western blot for Nef and internal protein Alix. (d) Quantification of the bands in panel C was conducted using an ImageJ application, with presented results illustrating the Nef/Alix ratio. Statistical analysis was carried out by unpaired Student's t test, and the p ‐value is displayed above the sample values. (e) EVs isolated from the conditioned media of HIV ADA ‐infected or uninfected MDM cell cultures, as well as EVs from the conditioned media of HEK293T cells transfected with Nef‐expressing (Nef EVs) or empty vector (Cont EVs), were equalised based on the protein content and applied to an ELISA plate coated with the anti‐Nef monoclonal antibody. The signal was revealed using the anti‐CD63 polyclonal antibody, followed by an HRP‐conjugated secondary antibody. The bars in the graph represent the mean ± SEM of the OD 450 ‐ OD 540 difference, with individual points indicating technical replicates. Statistical analysis was performed using ordinary one‐way ANOVA with Sidak correction for multiple comparisons. (f) Bodipy‐labelled EVs were captured by magnetic nanoparticles coupled with anti‐tetraspanin antibodies (anti‐CD63, anti‐CD81 and anti‐CD9), and the EV surface was stained for the presence of Nef with AF647‐labelled anti‐Nef antibody (left panel). Staining of the surface Nef, along with intravesicular Nef staining, was performed with the same AF647‐labelled anti‐Nef‐ antibody after fixation and permeabilisation (right panel). A representative experiment of two is shown. (g, h) Control EVs and Nef‐RFP EVs, isolated from supernatants of transfected HEK293T cells, were analysed using two anti‐RFP antibodies, which were alternately utilised for capturing and detecting.

Article Snippet: Chips were washed three times and incubated with anti‐CD9‐CF488 (Abcam, cat. # ab267502), anti‐CD81‐CF555 (Bioss, cat. # BS‐6934R‐A555) and anti‐CD63‐CF647 (Abcam, cat. # ab233056) following the manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Produced, Western Blot, Isolation, Infection, Expressing, Staining, Control