draq5  (Novus Biologicals)

Journal: Journal of Functional Biomaterials

Article Title: Influence of Micro-Nanostructured Anatase-Coated SLA Titanium on Macrophage Behavior

doi: 10.3390/jfb17030111

Figure Lengend Snippet: Behavior of M0-, M1-, and M2-MDMs on differently modified titanium Surfaces. ( A ) Representative CLSM images showing M0-, M1-, and M2-MDMs on coverslip, Ti-machined, Ti-SLA, and Ti-SLA-anatase surfaces after 48 h of incubation. The cytoskeleton (F-actin) was stained with FITC-Phalloidin (green), and nuclei were stained with DRAQ5 (purple). ( B ) Representative SEM images of M0-, M1, and M2 macrophages on coverslip and titanium surface modifications. ( C ) Metabolic activity was assessed using the CCK-8 assay, with values normalized to coverslip M0 (set as 100%). Data are presented as mean ± SEM. n = 3. No significant differences were observed between groups (Friedman test with Dunn’s multiple comparisons test).

Article Snippet: Samples were blocked in 1% bovine serum albumin (BSA; VWR International, Leuven, Belgium; Cat No. 422361V) and 0.05% Tween-20 (Roth, Karlsruhe, Germany; Cat. No. 9127.1) in PBS for 30 min. Cytoskeletal staining was performed using 5 μg/mL phalloidin–FITC (Sigma Aldrich, Saint Louis, MI, USA; Cat. No. P5282) in blocking buffer for 1 h, followed by nuclear staining with 10 μM DRAQ5 (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat No. 130-117-344) for 15 min at RT.

Techniques: Modification, Incubation, Staining, Activity Assay, CCK-8 Assay

Journal: bioRxiv

Article Title: Cholesterol remodels the endoplasmic reticulum to control myofibroblastic CAF function

doi: 10.64898/2026.02.16.706237

Figure Lengend Snippet: (A) Measurement of the relative incorporation of [U- 14 C]glucose into protein, non-polar, RNA, DNA and polar fractions following 24 hours of labelling, n=3. (B) Relative incorporation of [1- 14 C]acetate into the lipid fraction of NFs and CAFs, n=3. (C) Gene Set Enrichment Analysis (GSEA) plot derived from RNA-Seq analysis of CAFs versus NFs, showing enrichment of a “lipid metabolic process” signature (GO:0006629). (D) Heatmap of cholesterol biosynthesis gene expression in NFs and CAFs, as determined by RNA-Seq analysis, n=3. (E) Quantification of relative total cholesterol levels in NFs and CAFs, n=3. (F) UMAP visualisation of 19,601 patient-derived breast tumour stromal cells analysed by single cell RNA-Seq (scRNA-Seq) from the Human Breast Cancer Atlas . Stromal clusters corresponding to immunomodulatory CAFs (iCAFs), myCAFs, perivascular (PVL) and endothelial cell subsets are indicated by colour (top). Feature plots illustrating expression of SQLE (bottom left) and SC5D (bottom right) in breast tumour stromal cell clusters. Gene expression is represented by log-normalised expression values. The myCAF cluster is delineated by a dashed line. (G) Representative filipin staining (green) of free cholesterol in NFs and CAFs. Nuclei are stained with Draq5 (blue). Dashed boxes indicate magnified regions shown to the right. White scale bar represents 10 µm, yellow scale bar represents 5 µm. (H) Quantification of relative cholesterol levels in the ER-enriched fraction isolated from NFs and CAFs, n=3.

Article Snippet: Filipin was removed, and cells were washed and imaged in PBS containing 5 μM Draq5 staining solution (Miltenyi Biotec) using a Zeiss Elyra PS.1 confocal microscope (Zeiss) equipped with a 63X/1.4 oil DIC objective.

Techniques: Derivative Assay, RNA Sequencing, Gene Expression, Single Cell, Expressing, Staining, Isolation