Journal: CNS Neuroscience & Therapeutics

Article Title: Endothelial Progenitor Cells Contribute to Neointima Formation in Rabbit Elastase‐Induced Aneurysm after Flow Diverter Treatment

doi: 10.1111/cns.12086

Figure Lengend Snippet: BM‐derived EPCs are tracked in neointima. In the early 2 weeks after autologous transfusion, EPCs with double fluorescence are observed in the subendothelium space (A–B) and around the stent struts(C–D). In the late 2 weeks after autologous transfusion, EPCs with double fluorescence are observed on the surface of neointima (E–H). Double‐positive cells were identified by showing yellow emission fluorescence for CFSE and blue emission fluorescence for Hoechst 33,342 concurrently.

Article Snippet: EPCs at the second to fourth passage were harvested at day 14, 17, and 20 of culture, double labeled with the DNA‐specific fluorochrome Hoechst 33,342 (1 μg/mL, Sigma, USA) for one hour and the intracellular fluorescent dye CFSE (carboxyfluorescein diacetate succinimidyl ester, 10 μmoL/mL, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 15 min at 37°C, suspended in 10 mL saline, and autologously transfused via the margin ear vein.

Techniques: Derivative Assay, Fluorescence

Journal: BMC Complementary and Alternative Medicine

Article Title: Dendritic cells pulsed with generated tumor cell lysate from Phyllanthus amarus Schum. & Thonn. induces anti-tumor immune response

doi: 10.1186/s12906-018-2296-4

Figure Lengend Snippet: The phagocytic capacity of dendritic cells towards CFSE-labeled tumor debris and FITC-dextran. a Flow cytometric analysis indicates the percentage of DCs population with positive uptake of CFSE stained tumor cell remnants in (a) iDCs, (b) HCT 116 treated-DCs and (c) MCF-7 treated-DCs. b Identification of DCs as high HLA-DR + population for (a) unloaded iDCs, (b) HCT 116-treated-DCs and (c) MCF-7-treated-DCs. c Representative FACs analysis data for FITC-dextran uptake by (a) iDCs, (b) HCT 116-TLY-pulsed-DCs, (c) MCF-7-TLY pulsed-DCs, and (d) LPS–stimulated only DCs. d The figure represents the statistically transformed data. ** Indicates significant difference (** P < 0.01) for TLY-treated-DCs versus iDCs. ## Indicated significant difference (## P < 0.01) of TLY-DCs versus LPS-stimulated DCs. The data are representative of three experiments comprising three different healthy donors and analyzed by one-way ANOVA followed by Dunnett’s test. iDCs, immature dendritic cell; TLY, tumor lysate; CFSE, Carboxy-fluorescein succinimidyl ester

Article Snippet: To validate this, both HCT 116 (colon cancer) and MCF-7 (breast cancer) cells were stained with a fluorescent dye CFSE [5, 6-carboxyfluorescein diacetate succinimidyl ester] (BD Pharmingen, BD Bioscience, USA) that gives a strong and stable green fluorescence.

Techniques: Labeling, Staining, Transformation Assay

Journal: The Journal of Experimental Medicine

Article Title: Cd8 + but Not Cd8 − Dendritic Cells Cross-Prime Cytotoxic T Cells in Vivo

doi:

Figure Lengend Snippet: CD8 + T cell proliferation in vivo after priming with OVA-loaded β2m −/ − (β2m 0/0 ) cells. Thy1.1 + CFSE-labeled OT-I cells were transferred into B6 mice. 3 d later, mice were primed by injection with irradiated OVA-loaded β2m −/ − cells. Spleen cells were isolated 3 d after priming and stained for Thy1.1, Vα2, and CD8. (A) CFSE profile of Thy1.1 + Vα2 + CD8 + OT-I cells from mice primed with OVA-loaded β2m −/ − cells. (B) The same profile from a mouse that received no OVA-loaded β2m −/ − cells.

Article Snippet: To label cells with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) the cells were resuspended at 5 × 10 7 cells/ml in PBS/0.1% BSA with 10 μM CFSE for 10 min at 37°C.

Techniques: In Vivo, Labeling, Injection, Irradiation, Isolation, Staining

Journal: Communications Biology

Article Title: Bacterial vitamin B6 is required for post-embryonic development in C. elegans

doi: 10.1038/s42003-024-05992-2

Figure Lengend Snippet: a Microscope images showing the distribution of GFP signal in the newly hatched L1 worms fed either unstained L. plantarum or CFSE-labeled fluorescent L. plantarum , 2 h after feeding. Scale bar, 50 μm. b Microscope images and bar graph demonstrating that newly hatched worms fed on L. plantarum without starvation were arrested at the early larval stage three days after being placed on the bacteria. Scale bar, 1000 μm. c Worms grown on L. plantarum for 6 days can recover to adults with viable progeny 4 days after switching to OP50 food. Scale bar, 1000 μm. d Microscope images and bar graph showing that dauer larvae fed L. plantarum had a similar percentage of adult worms to those fed on OP50, 2 days after being placed on the bacteria. Scale bar, 1000 μm. All data are representative of at least three independent experiments. n = number of worms scored. Data are represented as mean ± SEM. Significance determined by unpaired two-tailed Student’s t -test. * Indicates P -value < 0.05, ** indicates P -value < 0.01, N.S. indicates non-significant difference.

Article Snippet: The green fluorescent dye 5(6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE, MedChemExpress) was used to track the bacteria.

Techniques: Microscopy, Labeling, Bacteria, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Injectable Biomimetic Hydrogels as Tools for Efficient T Cell Expansion and Delivery

doi: 10.3389/fimmu.2018.02798

Figure Lengend Snippet: PIC gels form stable gels in vivo that do not cause inflammation. (A,B) Representative example (A) and quantification (B) of the radiant efficiency of IVIS imaging of mice s.c. injected with 100 μl Cy5-labeled 1.5 mg/mL PIC polymers mixed with T cells in the dorsal flank. N = 6–20 in three independent experiments. (C,D) The percentage of SSC high Ly6G + neutrophils (D) and CD11b + CD11c − macrophages (E) of all alive CD45.2 + cells that where surrounding or inside the PIC gel, compared to a similar region of skin in mice injected with medium and T cells. N = 7–9 in at least 3 independent experiments. (E) The percentage of Cy5 + cells of the respective subset surrounding or inside the PIC gel of mice injected with Cy5-labeled PIC gel. n = 7–9 in at least 3 independent experiments. (F) The percentage of Cy5 + cells of all CD11c + CD11b + DCs in the non-draining lymph node (ndLN) and draining lymph node (dLN) on day 7. n = 4 in 1 independent experiment. (G) The level of TNFα in serum 1 day after injecting mice with Cy5-labeled PIC polymers or medium mixed with T cells. (B–G) Values represent mean and SEM. (B) Data were analyzed with the Kruskal Wallis test and the Dunns post-test. ** p ≤ 0.01 (C–E) Data were analyzed with the a two-way ANOVA and Bonferroni post-test. (G) Data were analyzed with the Mann Whitney test. ns, not significant.

Article Snippet: Fluorescent images were collected with the IVIS Lumina imaging system (Cy5 signal: 640 nm, Cy5.5 filter, CFSE signal: 465 nm, GFP filter, Perkin Elmer) after 2 h, 1, 3, 7, 14, 21, or 28 days to investigate gel localization and cellular localization.

Techniques: In Vivo, Imaging, Injection, Labeling, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Injectable Biomimetic Hydrogels as Tools for Efficient T Cell Expansion and Delivery

doi: 10.3389/fimmu.2018.02798

Figure Lengend Snippet: T cells are encapsulated within PIC gels after in vivo gelation and are released into the blood and distant organs. (A) Representative IVIS fluorescent images of the radiant efficiency of a mouse injected s.c. with 100 ul Cy5-labeled 1.5 mg/mL PIC gel mixed with CFSE-labeled pan T cells in the dorsal flank. (B) Representative image of a 10 μm section of FFPE-treated PIC gel in the skin 1 day after injection. Scale bar = 400 um. (C) Representative quantification of the number of CD3 + T cells per mm 2 PIC gel of 10 um sections through the PIC gel with typically 20–30 um in between each section (1 day after injection). (D) Quantification of the average number of CD3 + T cells per mm 2 PIC gel of 10 um sections through the PIC gel ( n = 2 or 3 per timepoint in 2 independent experiments). Data analyzed with Kruskal Wallis test with dunn's post test relative to day 1, * p ≤ 0.05. (E) Setup of experiment to study release of T cells from PIC gels after injection. (F) Quantification of the percentage of CD45.1 + cells of all CD3 + T cells retrieved from PIC gels and surrounding skin. n = 5–7 in at least 3 independent experiments for timepoints day 1–21, n = 2 in 1 independent experiment for day 28. Data analyzed with Kruskal Wallis test with dunn's post test relative to day 1, not significant. (G) Representative flow cytometry plot demonstrating CD45.1 + cells gated on CD3 expression in the draining lymph node (LN) on day 3 after injection of PIC gel with CD45.1 + T cells. (H) Quantification of the percentage of CD45.1 + cells of all CD3 + T cells in the draining lymph node (LN), non-draining lymph node, spleen and blood. n = 7–9 in at least three independent experiments for timepoints day 1–21, n = 6 in one independent experiment for timepoint day 28. (D,F,H) Values represent mean and SEM.

Article Snippet: Fluorescent images were collected with the IVIS Lumina imaging system (Cy5 signal: 640 nm, Cy5.5 filter, CFSE signal: 465 nm, GFP filter, Perkin Elmer) after 2 h, 1, 3, 7, 14, 21, or 28 days to investigate gel localization and cellular localization.

Techniques: In Vivo, Injection, Labeling, Flow Cytometry, Expressing

Journal: Journal of the Chinese Medical Association : JCMA

Article Title: Phenylalanyl-tRNA synthetase subunit beta downregulation by spi1 proto-oncogene modulates lung adenocarcinoma progression and immune microenvironment via mammalian target of rapamycin pathway

doi: 10.1097/JCMA.0000000000001286

Figure Lengend Snippet: FARSB promotes tumorigenesis and anti-tumor immunity in LUAD. A, qRT-PCR analysis of FARSB mRNA levels in different cell groups (si-NC, control group; si-FARSB, FARSB knockdown group). B, WB analysis of FARSB protein expression in various cell groups. C, CCK-8 assay evaluating the effect of FARSB knockdown on cell viability. D, EdU assay assessing the impact of FARSB knockdown on cell proliferation. E and F, Flow cytometry analysis of the effect of FARSB knockdown on cell apoptosis. G, WB analysis of PD-L1 (an immune checkpoint protein) expression levels. H, CFSE fluorescence staining assessing CD8 + T cell proliferation (CFSE dye dilution measures CD8 + T cell division). I-K, Flow cytometry analysis of IFN-γ (I), GZMB (J), and TNF-α (K) expressions in tumor-infiltrating CD8 + T cells; these cytokines reflect CD8 + T cell cytotoxic function. N = 3, *** p < 0.001 and **** p < 0.0001 denote statistical significance. CFSE = carboxyfluorescein succinimidyl ester; FARSB = phenylalanyl-tRNA synthetase subunit beta; GZMB = Granzyme B; IFN-γ = interferon-gamma; LUAD = lung adenocarcinoma; PD-L1 = programmed death-ligand 1; TNF-α = tumor necrosis factor-alpha; WB = western blot.

Article Snippet: Carboxyfluorescein succinimidyl ester (CFSE) dye (E-CK-A345, Elabscience, China) was diluted to the appropriate concentration and added to the CD8 + T cell suspension.

Techniques: Quantitative RT-PCR, Control, Knockdown, Expressing, CCK-8 Assay, EdU Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: Journal of the Chinese Medical Association : JCMA

Article Title: Phenylalanyl-tRNA synthetase subunit beta downregulation by spi1 proto-oncogene modulates lung adenocarcinoma progression and immune microenvironment via mammalian target of rapamycin pathway

doi: 10.1097/JCMA.0000000000001286

Figure Lengend Snippet: FARSB Activates the mTOR signaling pathway, promoting LUAD tumorigenesis and anti-tumor immunity. A, GSEA analysis of enriched pathways associated with differential FARSB expression in LUAD. B, WB analysis of the expression of the pathway-related proteins p-mTOR, mTOR, p-p70S6K, p70S6K, p-4EBP1, and 4EBP1. C, CCK-8 assays evaluating cell viability. D, EdU assays assessing cell proliferation. E, Flow cytometry analysis of cell apoptosis. F, WB analysis of PD-L1 expression levels. G, CFSE fluorescence staining assessing CD8 + T cell proliferation. H-J, Flow cytometry analysis of IFN-γ (H), GZMB (I), and TNF-α (J) expressions in tumor-infiltrating CD8 + T cells; these cytokines reflect CD8 + T cell cytotoxic function. n = 3, ** p < 0.01, *** p < 0.001, **** p < 0.0001 denote statistical significance. CFSE = carboxyfluorescein succinimidyl ester; FARSB = phenylalanyl-tRNA synthetase subunit beta; GZMB = Granzyme B; IFN-γ = interferon-gamma; LUAD = lung adenocarcinoma; mTOR = mammalian target of rapamycin; ns = no significant differences; PD-L1 = programmed death-ligand 1; TNF-α = tumor necrosis factor alpha; WB = western blot.

Article Snippet: Carboxyfluorescein succinimidyl ester (CFSE) dye (E-CK-A345, Elabscience, China) was diluted to the appropriate concentration and added to the CD8 + T cell suspension.

Techniques: Expressing, CCK-8 Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: Journal of the Chinese Medical Association : JCMA

Article Title: Phenylalanyl-tRNA synthetase subunit beta downregulation by spi1 proto-oncogene modulates lung adenocarcinoma progression and immune microenvironment via mammalian target of rapamycin pathway

doi: 10.1097/JCMA.0000000000001286

Figure Lengend Snippet: SPI1 Downregulates FARSB to modulate the mTOR signaling pathway, thus influencing LUAD progression and anti-tumor immunity. A, qRT-PCR analysis of FARSB and SPI1 mRNA levels in different cell groups. B, WB analysis of FARSB and SPI1 protein expression. C and D, WB analysis of mTOR signaling pathway-related proteins. E, CCK-8 assays evaluating cell viability. F, EdU assays assessing cell proliferation. G, Flow cytometry analysis of cell apoptosis. H, WB analysis of PD-L1 expression levels. I: CFSE fluorescence staining assessing CD8 + T cell proliferation. J-L: Flow cytometry analysis of IFN-γ (J), GZMB (K), and TNF-α (L) expressions in tumor-infiltrating CD8 + T cells; these cytokines reflect CD8+ T cell cytotoxic function. n = 3, *** p < 0.001, **** p < 0.0001 denote statistical significance. FARSB = phenylalanyl-tRNA synthetase subunit beta; GZMB = Granzyme B; IFN-γ = interferon-gamma; LUAD = lung adenocarcinoma; mTOR = Mammalian target of rapamycin; ns = no significant differences or significant correlation.; PD-L1 = programmed death-ligand 1; qRT-PCR = quantitative reverse transcription-polymerase chain reaction; TNF-α = tumor necrosis factor alpha; WB = western blot.

Article Snippet: Carboxyfluorescein succinimidyl ester (CFSE) dye (E-CK-A345, Elabscience, China) was diluted to the appropriate concentration and added to the CD8 + T cell suspension.

Techniques: Quantitative RT-PCR, Expressing, CCK-8 Assay, Flow Cytometry, Fluorescence, Staining, Reverse Transcription, Polymerase Chain Reaction, Western Blot