sucrose buffer b  (Biotium)

Journal: Journal of Extracellular Vesicles

Article Title: M1 Macrophage‐Derived Small Extracellular Vesicles as Synergistic Nanotherapeutics: Harnessing Intrinsic Anticancer Activity and Drug Delivery Capacity

doi: 10.1002/jev2.70242

Figure Lengend Snippet: Comprehensive sEV characterization and M1‐sEV Immune Evasion. (a) Fluorescence‐activated cell sorting (FACS) analysis of CD86 (M1 marker) and CD206 (M2 marker) in lipopolysaccharide (LPS; 500 ng/mL)‐stimulated M0 macrophages to confirm M1 polarization prior to sEV isolation. Transmission electron microscopy (TEM) images showing spherical nanostructures and corresponding size distribution analysis ( n = 80) of (b) 3T3‐sEVs, (c) M0‐sEVs, and (d) M1‐sEVs. (e) Western blot analysis of sEV markers and immunomodulatory ligand. Nanoparticle tracking analysis (NTA) showing size distribution and particle concentration profiles ( n = 5) of (f) 3T3‐sEVs, (g) M0‐sEVs, and (h) M1‐sEVs. Representative ex vivo fluorescence images and corresponding biodistribution quantification ( n = 3, mean ± s.d.) of major organs resected from mice administered with (i, m) Ctrl (PBS), (j, n) 3T3‐sEVs, (k, o) M0‐sEVs, and (l, p) M1‐sEVs. DiD‐labelled sEVs (measurement: ex/em = 605/670 nm). Quantification normalized to the fluorescence signal intensity of DiD‐3T3‐sEVs. One‐way ANOVA with Tukey's post‐test. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Fluorescence‐activated cell sorting system (FACS; Cytek Northern LightsTM (NL)‐CLC NL‐2000; Cytek Biosciences, CA, USA) was used to sort and detect the cells.

Techniques: Fluorescence, FACS, Marker, Isolation, Transmission Assay, Electron Microscopy, Western Blot, Concentration Assay, Ex Vivo

Journal: Journal of Extracellular Vesicles

Article Title: M1 Macrophage‐Derived Small Extracellular Vesicles as Synergistic Nanotherapeutics: Harnessing Intrinsic Anticancer Activity and Drug Delivery Capacity

doi: 10.1002/jev2.70242

Figure Lengend Snippet: Tumour‐homing properties of M1‐sEVs. (a) Mass spectrometry (MS)‐based proteomic profiling of sEVs. Protein accession numbers from UniProtKB/Swiss‐Prot; black boxes with [+]: detected proteins; white boxes with [−]: undetected protein; Gal‐3: Galectin‐3; VEGFR1: Vascular endothelial growth factor receptor‐1; NRP: Neuropilin; HSP: Heat shock protein; CHC: Clathrin heavy chain; CLC: Clathrin light chain. Fluorescence‐activated cell sorting (FACS) analysis of DiD‐labelled sEV uptake to MCF‐7 cells at (b) 1 h, (c) 4 h, and (d) 8 h at 37°C, with (f) corresponding quantification ( n = 3, mean ± s.d.). (e) FACS analysis of temperature‐dependent uptake at 4°C, with (g) corresponding quantification ( n = 3, mean ± s.d.). Quantification threshold: DiD fluorescence intensity > 10 4 . (h) Two‐photon confocal laser scanning microscopy (TP‐CLSM) images of DiD‐labeled sEV uptake in MCF‐7 spheroids in inverted colloidal crystal (iCC) framework (Blue: live cells; Green: 5‐DTAF‐labeled iCC; Red: DiD‐labelled sEV). (i) In vivo fluorescence imaging of DiD‐sEVs at various time points following tail‐vein intravenous (i.v.) administration in MCF‐7 tumour–bearing female NCr nude mice. (j) Quantitatively collected DiD‐sEV intensity in the spheroid penetration depth in TP‐CLSM images ( N > 30). (k) Time‐dependent quantification of DiD‐sEV accumulation in tumour based on in vivo systemic biodistribution imaging ( n = 4, mean ± s.d.). One‐way ANOVA with Tukey's post‐test. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Fluorescence‐activated cell sorting system (FACS; Cytek Northern LightsTM (NL)‐CLC NL‐2000; Cytek Biosciences, CA, USA) was used to sort and detect the cells.

Techniques: Mass Spectrometry, Fluorescence, FACS, Confocal Laser Scanning Microscopy, Labeling, In Vivo, Imaging

Journal: Cell reports

Article Title: PI5P4Kα promotes glucose and iron acquisition to support metabolic fitness in pancreatic cancer

doi: 10.1016/j.celrep.2025.116199

Figure Lengend Snippet: (A) FerroOrange staining in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2. Immunofluorescence microscopy images of FerroOrange and DAPI indicating the representative effect of three independent experiments (scale bar, 20 μm) and quantitation of immunofluorescence microscopy images. Data are presented as signal intensity relative to the sh scr condition and are the average of three independent experiments. (B) Transferrin uptake in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2. Data are presented as signal intensity relative to the sh scr condition and are the average of three independent experiments. (C) Surface staining of CD71 quantified using flow cytometry. Data are presented as median intensity relative to the sh scr condition and are the average of three independent experiments. (D) FerroOrange staining in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2 in the presence of FAC. (E) Quantification of relative cell number in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2 in the absence or presence of ferrostatin, ferrostatin with ferric ammonium citrate (FAC), and FAC alone. Data are presented relative to the sh scr condition for each respective treatment and are the average of three independent experiments. (F) Correlation plots between the expression of PIP4K2A from TCGA, with the indicated gene signatures. (G) Kaplan-Meier overall survival curve of patients with PDAC stratified by the expression levels of genes in the indicated gene signatures. (H–J) The MIA PaCa-2 cells were transduced with sh scr, sh α#1, or sh α#2 in the absence or presence of FAC. (H) Glucose consumption. (I) Lactate production. Data are presented relative to the cell number for each respective treatment and are representative of three independent experiments. (J) Immunoblot assessing GLUT1 and PI5P4Kα levels. Tubulin was used as a loading control. Data are representative of three independent experiments. (B–E, H, and I) Data are presented as mean ± SD. Statistical significance was calculated using unpaired two-tailed Student’s t test without (E, H, and I) or with (B–D) Welch’s correction and with pairwise Wilcoxon rank-sum test (F). ns, not significant, * p < 0.05, ** p ≤ 0.001, and *** p ≤ 0.0001.

Article Snippet: The following reagents were used: Polybrene (NC9840454, Santa Cruz Biotechnology), 37% Formaldehyde (RSOF0010, RICCA chemical company), Crystal Violet (C0775, Sigma), Emricasan (510230, MedKoo Biosciences), Tetramethylrhodamine Ethyl Ester Perchlorate (TMRE) (T669, Thermo Fisher Scientific), Human Transferrin, CF594 Conjugate (00084, Biotium), DAPI (422801, BioLegend), Hoechst 33342 (0219030525, MP Biomedicals), Doxycycline hyclate (D9891, Sigma).

Techniques: Staining, Transduction, Immunofluorescence, Microscopy, Quantitation Assay, Flow Cytometry, Expressing, Western Blot, Control, Two Tailed Test

Journal: Cell reports

Article Title: PI5P4Kα promotes glucose and iron acquisition to support metabolic fitness in pancreatic cancer

doi: 10.1016/j.celrep.2025.116199

Figure Lengend Snippet: (A) FerroOrange staining in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2. Immunofluorescence microscopy images of FerroOrange and DAPI indicating the representative effect of three independent experiments (scale bar, 20 μm) and quantitation of immunofluorescence microscopy images. Data are presented as signal intensity relative to the sh scr condition and are the average of three independent experiments. (B) Transferrin uptake in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2. Data are presented as signal intensity relative to the sh scr condition and are the average of three independent experiments. (C) Surface staining of CD71 quantified using flow cytometry. Data are presented as median intensity relative to the sh scr condition and are the average of three independent experiments. (D) FerroOrange staining in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2 in the presence of FAC. (E) Quantification of relative cell number in the indicated cell lines transduced with sh scr, sh α#1, or sh α#2 in the absence or presence of ferrostatin, ferrostatin with ferric ammonium citrate (FAC), and FAC alone. Data are presented relative to the sh scr condition for each respective treatment and are the average of three independent experiments. (F) Correlation plots between the expression of PIP4K2A from TCGA, with the indicated gene signatures. (G) Kaplan-Meier overall survival curve of patients with PDAC stratified by the expression levels of genes in the indicated gene signatures. (H–J) The MIA PaCa-2 cells were transduced with sh scr, sh α#1, or sh α#2 in the absence or presence of FAC. (H) Glucose consumption. (I) Lactate production. Data are presented relative to the cell number for each respective treatment and are representative of three independent experiments. (J) Immunoblot assessing GLUT1 and PI5P4Kα levels. Tubulin was used as a loading control. Data are representative of three independent experiments. (B–E, H, and I) Data are presented as mean ± SD. Statistical significance was calculated using unpaired two-tailed Student’s t test without (E, H, and I) or with (B–D) Welch’s correction and with pairwise Wilcoxon rank-sum test (F). ns, not significant, * p < 0.05, ** p ≤ 0.001, and *** p ≤ 0.0001.

Article Snippet: Transferrin (human) CF ® 594 Conjugate , Biotium , Cat# #00084.

Techniques: Staining, Transduction, Immunofluorescence, Microscopy, Quantitation Assay, Flow Cytometry, Expressing, Western Blot, Control, Two Tailed Test