b16f10 cells  (Revvity)

Journal: Science Advances

Article Title: Eyedrop-based macromolecular ophthalmic drug delivery for ocular fundus disease treatment

doi: 10.1126/sciadv.abq3104

Figure Lengend Snippet: ( A ) Immunofluorescence imaging indicating the distribution of tight junction–related proteins, including occludin and E-cadherin, after different treatments (scale bars, 10 μm). ( B to D ) Western blotting images showing the expression of occludin, E-cadherin, MLC, and p-MLC and statistical analysis of MLCs and p-MLC in HCEC monolayers after incubation with different nanocomplexes. ns, not significant. ( E ) TEER of HCECs after different treatments for 1 hour. ( F ) Schematic showing the in vitro simulated corneal epithelial barrier consisting of an HCEC monolayer in the upper chamber and PDL1-expressing B16F10 cells in the lower chamber of a Transwell. ( G ) Intensity of fluorescence-labeled aPDL1 that combined with the remaining unblocked PDL1 antigen expressed in B16F10 cells. ( H ) TEM images of rabbit corneal epithelial tissue after treatment with PBS, SDS, FCS, or CS (scale bars, 5 μm) and magnified TEM images (scale bar, 500 nm). ( I ) Confocal images of mouse eyeballs 6 hours after FCS-FITC/IgG-Cy5.5, CS-FITC/IgG-Cy5.5, or free IgG-Cy5.5 eyedrops were applied. The cornea, iris, and retina were partially magnified (scale bars, 500 μm). Data were represented as means ± SD. P values in (D) and (G) were calculated by using t test (* P < 0.05).

Article Snippet: Then, B16F10 cells were collected, and the penetrated anti-PDL1 bound to PDL1 expressed on B16F10 cells was detected using a competitive method by flow cytometry (aPDL1-PE, BioLegend, catalog no. 124308).

Techniques: Immunofluorescence, Imaging, Western Blot, Expressing, Incubation, In Vitro, Fluorescence, Labeling

Journal: Science Advances

Article Title: Eyedrop-based macromolecular ophthalmic drug delivery for ocular fundus disease treatment

doi: 10.1126/sciadv.abq3104

Figure Lengend Snippet: ( A ) Schematic illustration of establishing choroidal melanoma and the design of animal experiments. The experimental groups included (i) untreated, (ii) intravenous injection of anti-PDL1, (iii) anti-PDL1 eyedrops, (iv) FCS eyedrops, and (v) FCS/anti-PDL1 eyedrops. ( B ) Representative in vivo bioluminescence images of mice with Luc-B16F10 choroidal melanoma after different treatments (scale bar, 1 cm). ( C ) Average bioluminescence signal intensities from tumors after different treatments. ( D ) Cumulative ophthalmorrhexis-free survival of mice in different groups. ( E ) Cumulative modality-free survival rate of mice in different groups. ( F ) Representative fluorescence-activated cell sorting analysis of CD8 + and CD4 + T cells in eyes collected from mice after different treatments. ( G to I ) Percentages of CD4 + T cells, CD8 + T cells in CD3 + cells, and GranzB + T cells in CD3 + CD8 + cells analyzed by flow cytometry. ( J ) Concentration of IFN-γ in eyes after different treatments evaluated by ELISA. ( K ) Representative immunofluorescence images indicating the infiltration of CD8 + T cells around choroidal melanoma (scale bar, 50 μm). ( L ) Representative H&E staining slices of mouse eyes with choroidal melanoma in different groups (scale bar, 500 μm). Data were represented as means ± SD. P values in (C) and (G) to (J) were calculated by using one-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001). P values in (D) and (E) were calculated by using unpaired t test (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: Then, B16F10 cells were collected, and the penetrated anti-PDL1 bound to PDL1 expressed on B16F10 cells was detected using a competitive method by flow cytometry (aPDL1-PE, BioLegend, catalog no. 124308).

Techniques: Injection, In Vivo, Fluorescence, FACS, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8 + T-cell infiltration into tumors

doi: 10.1038/s41467-022-31713-6

Figure Lengend Snippet: a Bar graphs showing the numbers of intratumor PD-1 + CD8 + TILs (normalized to tumor weights) isolated from the YUMMER1.7 tumors expressing different HRS variants ( n = 5). b Percentages of Ki-67 + (left) and Granzyme B + (right) cells of PD-1 + CD8 + T cells in YUMMER1.7 tumors ( n = 5). c Growth of different HRS-expressing YUMMER1.7 tumors in C57BL/6 mice treated with anti-PD-1 or isotype control antibodies ( n = 5). d – f Growth of B16F10 tumors expressing HRS WT ( d , left), HRS S345A ( e , left), or HRS S345D ( f , left) treated with vehicle, anti-PD-1, BVD-523 (BVD), or anti-PD-1 plus BVD-523 as indicated. The numbers of PD-1 + CD8 + TILs (normalized to tumor weights) were shown in right ( n = 6). The experiments were repeated three times independently with similar results obtained ( a – f ). Data represent mean ± s.d. Statistical analyses were performed using one-way ANOVA ( a , b , d right, e right, f right) or two-way ANOVA ( c , d left, e left, f left). Tukey’s test was used following ANOVA. Source data are provided as a Source Data file.

Article Snippet: Human peripheral CD8 + T cells (1 × 10 5 per well-96 well plate) obtained from Human Immunology Core of University of Pennsylvania or murine CD8 + T cells (1 × 10 5 per well-96 well plate) purified from splenocytes and lymphocytes using EasyJet Mouse CD8 + T-cell Isolation Kit (STEMCELL) were stimulated with anti-CD3 (2 μg/ml, Biolegend) and anti-CD28 (2 μg/ml, Biolegend) antibodies for 24 hr and then incubated with indicated WM9 cell/xenograft-derived sEVs or B16F10 cell/xenograft-derived sEVs (20 μg/ml) with or without PD-L1 blocking for 48 hr in the presence of anti-CD3 and CD28 antibodies.

Techniques: Isolation, Expressing

Journal: Nature Communications

Article Title: HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8 + T-cell infiltration into tumors

doi: 10.1038/s41467-022-31713-6

Figure Lengend Snippet: a – b Growth of PD-L1 -KO B16F10 tumors in C57BL/6 and Rag2 −/− mice with indicated sEV treatments ( n = 6 for each group). c Scatter-bar graphs indicating the number of intratumor PD-1 + CD8 + TILs (normalized to tumor weights) in B16F10 tumors expressing HRS mutants in ( a ). d , e Percentages of Ki-67 + and Granzyme B + cells in human CD8 + T cells treated with indicated sEVs ( n = 5). f Heatmap of RPPA data showing the significantly changed proteins in peripheral CD8 + cells with indicated sEVs treatment. Proteins considered significantly changed between vehicle control and sEV WT treatment group are described in Supplementary Fig. . g Cytotoxicity elicited by mouse splenocytes with or without exposure to indicated B16F10 cell-derived sEVs ( n = 5). See METHODS for details. h Schema for sEV co-xenograft system (left) (See details in METHODS ). CD8 + T cells were transferred into Rag2 −/− mice 2 days before tumor injection. PD-L1 -KO B16F10 cells were mixed with Matrigel (ECM) and sEVs from B16F10 cells and subcutaneously inject into the mice. The number of PD-1 + CD8 + T cells in the co-grafted tumors was determined on Day 12 ( n = 5) (right). i Schema for the chemotaxis assay to access CD8 + T cells migration with sEVs and fibronectin (ECM) (left) (See the details in METHODS ). Percentages of transmigrated human CD8 + T cells stimulated with CXCL9 (100 ng/mL) in chemotaxis assay. 3-μm size pores were blocked by fibronectin with vehicle or indicated WM9 cell-derived sEVs ( n = 5) (right). The experiments were repeated three ( a – c , h ) and five ( d , e , g , i ) times independently with similar results obtained. Data represent mean ± s.d. Statistical analyses were performed using one-way ANOVA ( c – e , g – i ) or two-way ANOVA ( a , b ). Tukey’s test was used following ANOVA. Source data are provided as a Source Data file.

Article Snippet: Human peripheral CD8 + T cells (1 × 10 5 per well-96 well plate) obtained from Human Immunology Core of University of Pennsylvania or murine CD8 + T cells (1 × 10 5 per well-96 well plate) purified from splenocytes and lymphocytes using EasyJet Mouse CD8 + T-cell Isolation Kit (STEMCELL) were stimulated with anti-CD3 (2 μg/ml, Biolegend) and anti-CD28 (2 μg/ml, Biolegend) antibodies for 24 hr and then incubated with indicated WM9 cell/xenograft-derived sEVs or B16F10 cell/xenograft-derived sEVs (20 μg/ml) with or without PD-L1 blocking for 48 hr in the presence of anti-CD3 and CD28 antibodies.

Techniques: Expressing, Derivative Assay, Injection, Chemotaxis Assay, Migration

Journal: Nature Communications

Article Title: HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8 + T-cell infiltration into tumors

doi: 10.1038/s41467-022-31713-6

Figure Lengend Snippet: a Cytotoxicity elicited by primed mouse splenocytes treated with or without sEVs derived from PD-L1 -KO B16F10 cells expressing indicated HRS mutants ( n = 5). b Cytotoxicity elicited by primed splenocytes treated with or without sEVs derived from B16F10 cells expressing indicated HRS mutants. sEVs were pretreated with anti-PD-L1 blocking antibody or isotype antibody controls ( n = 5). c The growth of PD-L1 -KO B16F10 tumors in C57BL/6 treated with indicated sEVs from B16F10 cells expressing various HRS mutants. The sEVs were preincubated with isotype control or anti-PD-L1 antibodies ( n = 6). d The number of intratumor PD-1 + CD8 + T cells (normalized to tumor weight) in tumors in ( c ). e Experimental schema for adoptive CD45.1 + and CD45.2 + CD8 + T cells co-transfer system (left). Number of intratumor PD-1 + CD8 + T cells (normalized to tumor weight) pre-treated with indicated sEVs blocked by isotype (CD45.1 + group) control or anti-PD-L1 (CD45.2 + group) (normalized to tumor weight) (right). n = 5. f Transwells were pretreated with fibronectin and sEVs from B16F10 cells with or without BVD-523. Percentages of transmigrated mouse CD8 + T cells induced by CXCL9 were accessed ( n = 5). g Cytotoxicity elicited by primed mouse splenocytes treated with sEVs derived from B16F10 cells with or without BVD-523 treatment ( n = 5). h Number of intratumor PD-1 + CD8 + T cells (normalized to tumor weight) in tumor co-grafted by the mixture including indicated sEVs, ECM (Matrigel) and PD-L1 -KO B16F10 cells ( n = 5). The experiments were repeated three ( b – h ) and five ( a ) times independently with similar results obtained. Data represent mean ± s.d. Statistical analyses were performed using one-way ANOVA ( a ) or two-way ANOVA ( b – h ). Tukey’s test ( a , c ) and Sidak’s ( b , d – h ) were used following ANOVA. Source data are provided as a Source Data file.

Article Snippet: Human peripheral CD8 + T cells (1 × 10 5 per well-96 well plate) obtained from Human Immunology Core of University of Pennsylvania or murine CD8 + T cells (1 × 10 5 per well-96 well plate) purified from splenocytes and lymphocytes using EasyJet Mouse CD8 + T-cell Isolation Kit (STEMCELL) were stimulated with anti-CD3 (2 μg/ml, Biolegend) and anti-CD28 (2 μg/ml, Biolegend) antibodies for 24 hr and then incubated with indicated WM9 cell/xenograft-derived sEVs or B16F10 cell/xenograft-derived sEVs (20 μg/ml) with or without PD-L1 blocking for 48 hr in the presence of anti-CD3 and CD28 antibodies.

Techniques: Derivative Assay, Expressing, Blocking Assay

Journal: Wiley Interdisciplinary Reviews. RNA

Article Title: microRNA ‐based diagnostic and therapeutic applications in cancer medicine

doi: 10.1002/wrna.1662

Figure Lengend Snippet: Genetic or viral modulation of miRNA activity in in vivo cancer models

Article Snippet: miR‐34a replacement , Dharmacon mimic , LPH nanoparticle with tumor targeting scFv , Lung metastasis, B16F10 melanoma cells , Skin (metastatic) , Inhibits tumor growth , Chen et al. ( ) .

Techniques: Activity Assay, In Vivo, Ex Vivo, Transduction, Expressing, Sequencing, Injection, Blocking Assay, Plasmid Preparation, Over Expression, CRISPR, Knock-Out, Irradiation

Journal: Wiley Interdisciplinary Reviews. RNA

Article Title: microRNA ‐based diagnostic and therapeutic applications in cancer medicine

doi: 10.1002/wrna.1662

Figure Lengend Snippet: Pharmacological modulation of miRNA activity in in vivo cancer models

Article Snippet: miR‐34a replacement , Dharmacon mimic , LPH nanoparticle with tumor targeting scFv , Lung metastasis, B16F10 melanoma cells , Skin (metastatic) , Inhibits tumor growth , Chen et al. ( ) .

Techniques: Activity Assay, In Vivo, Inhibition, Expressing, Injection, IV Injection, Modification, Conjugation Assay

Journal: Cell reports

Article Title: Chronic stress physically spares but functionally impairs innate-like invariant T cells

doi: 10.1016/j.celrep.2021.108979

Figure Lengend Snippet:

Article Snippet: Mouse: B16-F10-Red-FLuc (B16-FLuc) melanoma cells , PerkinElmer , Cat # BW124734.

Techniques: Control, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, In Situ, Selection, cDNA Synthesis, Software

Journal: Cell reports

Article Title: Chronic stress physically spares but functionally impairs innate-like invariant T cells

doi: 10.1016/j.celrep.2021.108979

Figure Lengend Snippet:

Article Snippet: Mouse: B16-F10-Red-FLuc (B16-FLuc) melanoma cells , PerkinElmer , Cat # BW124734.

Techniques: Control, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, In Situ, Selection, cDNA Synthesis, Software