elisa kits  (R&D Systems)

Journal: Advanced Science

Article Title: Intercellular Horizontal Transfer of TXNDC5 mRNA via Extracellular Vesicles Contributes to Tumor‐Associated Macrophage‐Mediated Prostate Cancer Metastasis

doi: 10.1002/advs.202511052

Figure Lengend Snippet: Primary lesions of mPCa exhibit increased M2 macrophage infiltration and a higher M2‐EVs/Ti‐EVs ratio compared to those of nmPCa. (A) Schematic diagram of the technical workflow to characterize M2 macrophages and M2 EVs in the primary lesions of PCa. (B) UMAP projection of all macrophages from the integrated scRNA‐seq dataset, colored by annotated cell subtypes: M1 macrophages, M2 macrophages, and Mixed macrophages. Cell numbers for each subset were indicated. (C) Analysis of the relative proportion of macrophage groups based on the integrated scRNA‐seq dataset in nmPCa and mPCa. (D) Association between the M2 phenotype and clinical outcome. M2 phenotype was defined by the expression of CD68 , CD163 , CD206 , IL10 , ARG1 , TGFB1 , VEGFA , and CCL22 . (E–G) Expression of M2 macrophage‐associated markers (represented by CD68, CD163, and CD206) in primary mPCa and nmPCa sites was shown by IHC (E, F) and IF(G). LR: low risk (patients with PSA value below 10 ng/mL, Gleason score below or equal to 7, and cT1‐cT2a disease); HR: high risk (patients with PSA value above 20 ng/mL, Gleason score above 7, cT2c‐cT4 disease, or a node‐positive disease). Scale bar, 50 µm. (H) Representative TEM images of Ti‐EVs from primary mPCa and nmPCa sites. Scale bar, 200 nm. (I) Size distribution of Ti‐EVs from primary mPCa and nmPCa sites showed by nFCM. (J) Western blotting of EV marker proteins (CD63, ALIX, and CD9) and contaminating protein (GM130). CL: cell lysate of tissue. (K, L) The proportions of CD68 + CD206 + EVs measured with nFCM in total Ti‐EVs from mPCa ( n = 5) compared to those from nmPCa ( n = 5). All experiments were repeated three times. Data presented as the mean ± SD. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; and ns for non‐significant data. Φ: macrophages.

Article Snippet: For the measurement of secretory protein concentrations, conditioned medium was collected and centrifuged for 20 min at 10 000 × g, and the supernatant was used to test the concentrations of TGF‐β1 (E‐EL‐0162, Elabscience), CCL22 (E‐EL‐H0029, Elabscience), and VEGFA (E‐EL‐H0111, Elabscience) with ELISA kits according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, Marker

Journal: Advanced Science

Article Title: Intercellular Horizontal Transfer of TXNDC5 mRNA via Extracellular Vesicles Contributes to Tumor‐Associated Macrophage‐Mediated Prostate Cancer Metastasis

doi: 10.1002/advs.202511052

Figure Lengend Snippet: CCM of M2 macrophages promotes migration and invasion of PCa cells. (A) Illustration of the strategy used to induce M0 and M2 macrophages in human leukemia monocytic THP‐1 cells. THP‐1 cells were differentiated into M0 macrophages by incubation with 100 ng/mL phorbol‐12‐myristate‐13‐acetate (PMA) for 48 h. M0 macrophages were polarized into M2 macrophages by culturing in 20 ng/mL IL‐4 and IL‐10 for 48 h. (B) Characterization of morphological changes in the course of differentiation from THP‐1 cells to M2 macrophages under a light microscope. Scale bars, 200 µm (100×), 100 µm (200×), 50 µm (400×). (C) ELISA revealed elevated levels of secretory TGF‐β, CCL22, and VEGFA in the CCM of M2 macrophages compared with those of M0 macrophages. (D) Evaluation of M2 macrophage‐associated protein markers by flow cytometry before and after differentiation. (E) Verification of classical M2‐associated genes by qRT‐PCR in M0 and M2 macrophages. Gene expression normalized to GAPDH . (F) The proportional change of CD68 + CD163 + cells upon induction was shown by IF. Scale bar, 100 µm. (G, H) Migration and invasion assays in M2 CCM‐treated versus M0 CCM‐treated DU145 (G) and PC3 (H) cells. (I, J) The wound healing assay showed different migration rates of DU145 (I) and PC3 (J) cells upon M2 CCM treatment. All experiments were repeated three times. Data presented as the mean ± SD. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; and ns for non‐significant data.

Article Snippet: For the measurement of secretory protein concentrations, conditioned medium was collected and centrifuged for 20 min at 10 000 × g, and the supernatant was used to test the concentrations of TGF‐β1 (E‐EL‐0162, Elabscience), CCL22 (E‐EL‐H0029, Elabscience), and VEGFA (E‐EL‐H0111, Elabscience) with ELISA kits according to the manufacturer's instructions.

Techniques: Migration, Incubation, Light Microscopy, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Wound Healing Assay