tirf microscope nikon eclipse ti inverted microscope  (Nikon)

Journal: iScience

Article Title: Neuroactive steroids activate membrane progesterone receptors to induce sex specific effects on protein kinase activity

doi: 10.1016/j.isci.2025.112352

Figure Lengend Snippet: Examining the effects of ORG on mPR cell surface stability (A) Representative images of GFP signal in cells imaged using TIRF microscopy following 20-min treatment with aCSF or 100nM mPR agonist, ORG. (B) Quantified and normalized fluorescence comparing baseline to ORG treatment for 20 min in mPRδ-GFP and mPRε-GFP cells. Data were normalized to aCSF treatment within each cell line. ∗∗∗ p < 0.001, N = 3, n = 9–12. Data are represented as mean ± SEM.

Article Snippet: The cells were placed in an imaging chamber heated to 37°C attached to a Nikon Eclipse Ti Inverted TIRF Microscope (Nikon Instruments).

Techniques: Microscopy, Fluorescence

Journal: Advanced Science

Article Title: Extracellular Vesicular Analysis of Glypican 1 mRNA and Protein for Pancreatic Cancer Diagnosis and Prognosis

doi: 10.1002/advs.202306373

Figure Lengend Snippet: Design of the ILN biochip assay for vesicular GPC1 expression. A) Schematic of the ILN biochip assay. Total Internal Reflection Fluorescence (TIRF) microscopy images were enlarged from 80 µm × 80 µm to 20 µm × 20 µm to show bright spots. B) GPC1 mRNA expression in EVs was verified by two MB designs recognizing different local sequences in GPC1 mRNA. One (2052‐2069, NM_002081.3) showed slightly better discrimination between 10 healthy individuals (Zen‐bio Inc.) and 10 PDAC patient samples (OSU) than the other (1173‐1195, NM_002081.3). C) The performance of MB (2052‐2069, (NM_002081.3) was confirmed by the serial dilution of synthetic standard vesicles with or without a synthetic RNA oligo as a GPC1 mRNA mimic. D) Transmission electron microscopy (TEM) images showed large and small EVs in a PDAC patient serum sorted by size exclusion chromatography (SEC, qEV column, 70 nm), Scale bar, 50 nm. E) NanoSight nanoparticle tracking analysis on EVs from conditioned media of PANC‐1 cells (Blue) or serum of a PDAC patient (Orange) showed two sized groups with mean diameters of 125 and 200 nm, respectively. The EV concentration was ranged in 1E7 particles per mL. F) PANC‐1cell‐derived EVs were captured by different antibodies and vesicular GPC1 mRNA expression was measured by the ILN biochip. Vesicular GPC1 mRNA showed higher expression in the exosome‐dominated EV subpopulation (captured by CD63, CD81, or CD9 antibody). G) Vesicular GPC1 protein showed higher expression in the EV subpopulations captured by PDAC‐associated antibodies (EpCAM/EGFR/GPC1). Calibration curves of EV GPC1 H) mRNA and I) mProtein expression in PANC‐1 cell‐derived EVs spiked into healthy donor serum in comparison with qRT‐PCR and ELISA, respectively. Both limit of detection (LOD) and cut‐off values to distinguish PDAC from control are marked. TFI; Total Fluorescence Intensity. Data were presented as means ± SD ( n = 2 wells, each well with 100 images). p values were determined by the two‐way ANOVA test. * p < 0.05.

Article Snippet: TIRF microscopy (Nikon Eclipse Ti Inverted Microscope System) was used to record and analyze sample images.

Techniques: Expressing, Fluorescence, Microscopy, Serial Dilution, Transmission Assay, Electron Microscopy, Size-exclusion Chromatography, Concentration Assay, Derivative Assay, Comparison, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: Advanced Science

Article Title: Extracellular Vesicular Analysis of Glypican 1 mRNA and Protein for Pancreatic Cancer Diagnosis and Prognosis

doi: 10.1002/advs.202306373

Figure Lengend Snippet: Distribution of GPC1 mRNA and protein in different EV subpopulations of non‐cancer and PDAC cells and human serum. A) Three sets of antibodies were used for capturing different EV subpopulations, including i) anti‐CD63/CD81/CD9 for exosome‐dominated vesicles (Exo), ii) anti‐ARF6/ANXA1 for microvesicle‐dominated vesicles (MV), and iii) anti‐EGFR/EpCAM/GPC1 for tumor‐associated microvesicle (tMV). B) Cell and vesicular GPC1 protein expression in each subpopulation (Exo, MV, and tMV) was measured by Western blot. Vesicular GPC1 protein is highly expressed in MV and tMV from the PANC‐1 cell line, but low in Exo. Lower GPC1 protein expression in a non‐cancerous cell line (HPDE6c7) and its EVs. C) GPC1 mRNA expression in cell, MV, tMV, and Exo were measured by qRT‐PCR. PANC‐1 and MIA PaCa‐2 cells expressed higher GPC1 mRNA expression in Exo but less in MV and tMV. D) By comparing qRT‐PCR results between two priming methods, random hexamer (Black, fragment) versus oligo dT (Gray, full length), we observed mostly GPC1 mRNA fragments rather than full length in Exo from PANC‐1 and MIA PaCa‐2 cells. Data were presented as means ± SD ( n = 3). E) GPC1 mRNA expression in Exo, MV, and tMV in HPDE6c7, PANC‐1, and MIA PaCa‐2 cell‐derived EVs using the ILN biochip assay. F) GPC1 mProtein expression in Exo, MV, and tMV in HPDE6c7, PANC‐1, and MIA PaCa‐2 cell‐derived EVs using the ILN biochip assay. TIRF microscopy images were enlarged from 80 µm × 80 µm to 20 µm × 20 µm to show bright spots. RFI: Relative Fluorescence Intensity. G) GPC1 mRNA and H) GPC1 mProtein expression in Exo, MV, and tMV in HD and PDAC patient samples ( n = 5). Data were presented as means ( n = 2 wells, each well with 100 images). p values were determined by the paired two‐tailed Student's t‐ test. * p < 0.05, ** p < 0.01, n.s., not significant.

Article Snippet: TIRF microscopy (Nikon Eclipse Ti Inverted Microscope System) was used to record and analyze sample images.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Random Hexamer, Derivative Assay, Microscopy, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Extracellular Vesicular Analysis of Glypican 1 mRNA and Protein for Pancreatic Cancer Diagnosis and Prognosis

doi: 10.1002/advs.202306373

Figure Lengend Snippet: The ILN biochip assay of GPC1 Exo‐mRNA and tMV‐mProtein expression for discovery, non‐blinded validation, and blinded validation studies. A,B) Representative TIRF images for GPC1 Exo‐mRNA and tMV‐mProtein expression. C) GPC1 Exo‐mRNA expression for discovery and non‐blinded validation studies. D) GPC1 tMV‐mProtein expression for discovery and non‐blinded validation studies. TIRF images were enlarged from 80 µm × 80 µm to 20 µm × 20 µm to show bright spots. E) Scatter plot for discovery samples from OSU. F) ROC curves of GPC1 Exo‐mRNA and tMV‐mProtein expression as a single‐ or dual‐marker for Stage I/II PDAC patient for discovery set compared to control. G) Scatter plot for non‐blinded validation samples from combined MSKCC and TVGH. H) ROC curves of dual GPC1 Exo‐mRNA and tMV‐mProtein expression for Stage I/II and Stage III/IV PDAC patients for non‐blinded validation set compared to control. I,J) GPC1 Exo‐mRNA and tMV‐mProtein expression for blinded validation samples. K) Scatter plot for blinded validation samples. L) Predicted ROC curves for dual‐GPC1 expression for blinded validation samples. Pairwise comparison p values were determined by the Mann–Whitney U test. * p < 0.05, *** p < 0.001, n.s., not significant. Dotted lines in (E, G, and K) indicate the cut‐off values from the control (green). All data were presented as means ( n = 2 wells, each well with 100 images).

Article Snippet: TIRF microscopy (Nikon Eclipse Ti Inverted Microscope System) was used to record and analyze sample images.

Techniques: Expressing, Biomarker Discovery, Marker, Control, Comparison, MANN-WHITNEY