flow cytometry attune nxt  (Thermo Fisher)

Journal: Frontiers in Immunology

Article Title: TREM1 is essential for maintaining stemness of liver cancer stem-like cells in hepatocellular carcinoma

doi: 10.3389/fimmu.2025.1618342

Figure Lengend Snippet: TREM1 knockout suppresses proliferation and migration while inducing apoptosis in HCC. (A) Western blot and (B) RT-PCR analysis confirms TREM1 knockout using CRISPR-Cas9 in Huh7 and HepG2 cell lines (n=3 per group). (C) Line graph shows CCK-8 assay assessing cell proliferation in control and TREM1 KO Huh7 and HepG2 cell lines (n=2–3 per group). (D) Cell migration in Huh7 and HepG2 cell lines, both control and TREM1 knockout groups assessed by in vitro transwell assay. Representative images of crystal violet staining captured at 24h. Number of migrated cells on each six-well plate counted in 3 independent experiments (n=3 per group) (E) Flow cytometry histogram plots depict Ki67-PI cell cycle analysis of control and TREM1 KO Huh7 and HepG2 cell lines. Representative plot from 3 independent experiments performed in triplicate (n=3 per group). (F) Flow cytometry analysis using Annexin V-PI staining shows significant increase in apoptosis during TREM1 silencing in Huh7 and HepG2 cell lines (n=3 per group). (G) RT2 PCR Array analysis of human apoptotic gene expression. Heatmap shows the expression of 84 key genes associated with apoptosis. Upregulated genes (red) and downregulated genes (blue) in Huh7 TREM1 KO cells shown. (H) Upregulation of pro-apoptotic genes and downregulation of anti-apoptotic genes in Huh7 TREM1 KO cells compared to the control group plotted using GraphPad Prism (version 9) (n=4–5 per group). ****p<0.0001.

Article Snippet: Following this the cells were acquired on the Attune NxT Acoustic Focusing flow cytometry platform (Thermo Fisher Scientific) and data were analyzed on FlowJo v10.0.

Techniques: Knock-Out, Migration, Western Blot, Reverse Transcription Polymerase Chain Reaction, CRISPR, CCK-8 Assay, Control, In Vitro, Transwell Assay, Staining, Flow Cytometry, Cell Cycle Assay, Gene Expression, Expressing

Journal: Frontiers in Immunology

Article Title: TREM1 is essential for maintaining stemness of liver cancer stem-like cells in hepatocellular carcinoma

doi: 10.3389/fimmu.2025.1618342

Figure Lengend Snippet: TREM1 promotes tumorigenicity, clonogenic potential and spheroid formation in CD133 + EpCAM + liver cancer stem-like cells. (A) Flow cytometry dot plots reveal significant TREM1 expression in CD133 + EpCAM + cells from Huh7, and HepG2 cell lines and HCC P1 patient sample. (B) RT-PCR analysis shows significant expression of stem cell factors in MACS-purified Huh7 CD133 + EpCAM + cells in comparison to CD133 - EpCAM - fractions (n=3 per group) (C) Flow cytometry dot plots depict reduction in CD133 + Epcam + LCSLCs during TREM1 silencing in Huh7 and HepG2 cells (n=4 per group). (D) RT-PCR analysis of MACS-purified LCSLCs reveals a significant decrease in stem cell factor expression during TREM1 ablation (n=3 per group). (E) Western blot analysis shows significant expression of stem cell proteins in MACS purified Huh7 and HepG2 control CD133 + EpCAM + cells in comparison to TREM1 KO CD133 + EpCAM + cells. (F) Spheroid formation assay shows TREM1 abrogation significantly limits spheroid formation and overall proliferation of LCSLCs. Keyence microscope was used for the acquisition of bright field images. Scale bars = 50 μm. Spheroids were counted using ImageJ (n=3 per group) (G) Colony formation assay demonstrates TREM1-positive LCSLCs form significantly more colonies than their KO counterparts. Representative images show TREM1 KO and Control Huh7 and HepG2 LCSLC colonies stained using crystal violet after 14 days. Data were plotted using GraphPad Prism (n=3 per group). (H) Representative images of tumors from NSG CDX models. 5000 Huh7 CD133 + EpCAM + LCSLCs from Control and TREM1 KO groups were injected subcutaneously (n=6 mice per group). The experiment was independently repeated three times for statistical analysis. ***p<0.001, ****p<0.0001.

Article Snippet: Following this the cells were acquired on the Attune NxT Acoustic Focusing flow cytometry platform (Thermo Fisher Scientific) and data were analyzed on FlowJo v10.0.

Techniques: Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Purification, Comparison, Western Blot, Control, Tube Formation Assay, Microscopy, Colony Assay, Staining, Injection

Journal: Frontiers in Immunology

Article Title: TREM1 is essential for maintaining stemness of liver cancer stem-like cells in hepatocellular carcinoma

doi: 10.3389/fimmu.2025.1618342

Figure Lengend Snippet: TREM1 inhibition via VJDT depletes LCSLCs, reduces tumor size, and decreases spheroid formation. (A) Tumor growth curves for Huh7 vehicle and VJDT treated mice (mean ± SEM, n=5 mice/group). Representative images of tumors from indicated groups on day 22. (B) Flow cytometry analysis shows a significant reduction in CD133 + EpCAM + LCSLCs in VJDT-treated tumors compared to the vehicle (n=4 per group). (C) Western blot analysis of two vehicle-treated and two VJDT-treated tumors shows reduced expression of stem cell-related proteins in VJDT-treated tumors. (D) Representative images from the spheroid formation assay demonstrate reduced spheroid formation following VJDT treatment. Scale bar = 50 µm. Spheroids were counted using ImageJ. **p<0.01, ***p<0.001, ns-not significant.

Article Snippet: Following this the cells were acquired on the Attune NxT Acoustic Focusing flow cytometry platform (Thermo Fisher Scientific) and data were analyzed on FlowJo v10.0.

Techniques: Inhibition, Flow Cytometry, Western Blot, Expressing, Tube Formation Assay