b16f10luc cells  (ATCC)

Journal: Scientific Reports

Article Title: Modeling pre-metastatic lymphvascular niche in the mouse ear sponge assay

doi: 10.1038/srep41494

Figure Lengend Snippet: The two major steps of the ear sponge assay are the sponge preparation ( a – f ) and the sponge implantation in mouse ears ( g – j ). ( a ) Sterile compressed gelatin sponges were cut with a sterile biopsy punch into small cylindrical pieces. ( b ) Sponges were next placed in a 96-well plate (one per well) with a forceps. ( c ) For each experiment, the positivity for the Luciferase gene expression in B16F10Luc+ transfected cells was checked by bioluminescence. Serial dilutions of cells, starting at 5 × 10 5 B16F10Luc+ cells (wells at left), were associated with proportional Xenogen signals. ( d ) A drop of the appropriate cell suspension (20 μl) was seeded on top of the sponge, allowing the progressive diffusion of the solution into the sponge during an incubation for 30 minutes at 37 °C. ( e ) Sponges were soaked with a collagen mix, placed immediately in a new well and incubated at 37 °C for 30 minutes to allow collagen gel polymerization. ( f ) Such collagen coating did not affect the bioluminescence emitted by cells when luciferin was added inside the sponge. In the picture, sponges soaked with 20 μl of 1 × 10 5 and 2 × 10 5 at left and right, respectively. ( g ) A horizontal incision was performed in the basal, external and central part of the mouse ear and the external mouse ear skin layer was smoothly detached from the cartilage with a thin forceps. ( h ) Sponges were placed in the incision. ( i ) The gelatin sponge was introduced inside the hole, between the external mouse skin layer and the cartilage. A suture point was made to close the skin incision. ( j ) The same procedure was repeated in the other mouse ear, using always sponges with the same experimental condition in the same mouse.

Article Snippet: Murine B16F10Luc+ melanoma cells expressing luciferase (Luc+) (Caliper Life Sciences, Hopkinton, MA) were used in this study.

Techniques: Sterility, Luciferase, Gene Expression, Transfection, Suspension, Diffusion-based Assay, Incubation

Journal: Scientific Reports

Article Title: Modeling pre-metastatic lymphvascular niche in the mouse ear sponge assay

doi: 10.1038/srep41494

Figure Lengend Snippet: The B16F10Luc+ tumor cell suspension (5 × 10 5 cells/50 μl) was directly injected between the two ear skin layers of C57BL/6J mice (Intradermal Injection) or added on a cylindrical piece of sponge and then implanted in mouse ears (Sponge Implantation). ( a ) At different time points (Day 2, 4, 9 and 14) bioluminescence in mouse ears was recorded in vivo with a Xenogen system. ( b ) The draining sentinel LNs were dissected and their bioluminescence was visualized ex vivo over time. Numeric values displayed in the color scale for radiance are expressed in photons/sec/cm 2 . “L” represents left ears/LNs and “R” represents right ears/LNs, n = 8 sponges or LNs per group (4 mice/group).

Article Snippet: Murine B16F10Luc+ melanoma cells expressing luciferase (Luc+) (Caliper Life Sciences, Hopkinton, MA) were used in this study.

Techniques: Suspension, Injection, In Vivo, Ex Vivo

Journal: Scientific Reports

Article Title: Modeling pre-metastatic lymphvascular niche in the mouse ear sponge assay

doi: 10.1038/srep41494

Figure Lengend Snippet: Sponges soaked with serum-free DMEM medium (control condition without tumor cells), 1.5 × 10 5 or 2 × 10 5 B16F10Luc+ cells diluted in serum-free DMEM medium, were implanted into both C57BL/6J mouse ears and mice were kept for 2 or 4 weeks. ( a ) Representative pictures showing the in vivo bioluminescent signals of the primary tumors developed in ears, at 2 (left panels) and 4 (right panels) weeks post-sponge insertion. Numeric values depicted in the color scale for the radiance are expressed in photons/sec/cm 2 . ( b ) Quantification of bioluminescent signal radiance expressed in photons/sec/cm 2 and measures of tumor sizes from histological sections of ear sponges. Data are presented as mean ± SEM, Wilcoxon-Mann-Whitney significance test was used to compare the mean parameter values, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 12 sponges per group (6 mice/group). ( c ) Representative pictures of ex vivo bioluminescent signal of the draining sentinel LNs at week 2 (left panels) and 4 (right panels) post-sponge implantation. Numeric values displayed in the color bar for radiance are expressed in photons/sec/cm 2 . For positive LNs, a bright field picture of LN is shown below the bioluminescent images. ( d ) Table of positive LNs incidence at week 2 and 4 post-sponge insertion. “L” represents left mouse ear/LN and “R” refers to right mouse ear/LN, n = 12 LNs per group (6 mice/group).

Article Snippet: Murine B16F10Luc+ melanoma cells expressing luciferase (Luc+) (Caliper Life Sciences, Hopkinton, MA) were used in this study.

Techniques: Control, In Vivo, MANN-WHITNEY, Ex Vivo

Journal: Scientific Reports

Article Title: Modeling pre-metastatic lymphvascular niche in the mouse ear sponge assay

doi: 10.1038/srep41494

Figure Lengend Snippet: Control sponges devoid of tumor cells (control) and sponges populated with 1.5 × 10 5 or 2 × 10 5 B16F10Luc+ tumor cells were implanted in C57BL/6J mouse ears for 2 ( a ) or 4 ( b ) weeks. Lymphatic initial vessels (LYVE-1 positivity, blue staining) and tumor cells (tyrosinase expression, pink staining) were detected by immunohistochemistry, in control ears (sponges soaked with serum-free DMEM without tumor cells) and in ear-bearing primary tumors. Representative pictures are belonging to the central area of the ear sponge. Lower panels in “a” and “b” represent a higher magnification of the region delineated by the square in the upper pictures. Black arrows indicate some representative lymphatic vessels and the dotted black lines in “b” delineate the well-established tumor areas. ESL and ISL indicate the external and internal skin layers in mouse ears, respectively. Asterisk indicates the cartilage in mouse ears. Scale bars represent 500 and 100 μm in the low and high magnification pictures, respectively.

Article Snippet: Murine B16F10Luc+ melanoma cells expressing luciferase (Luc+) (Caliper Life Sciences, Hopkinton, MA) were used in this study.

Techniques: Control, Staining, Expressing, Immunohistochemistry

Journal: Scientific Reports

Article Title: Modeling pre-metastatic lymphvascular niche in the mouse ear sponge assay

doi: 10.1038/srep41494

Figure Lengend Snippet: Sponges devoid of tumor cells (controls) and sponges populated with 1.5 × 10 5 or 2 × 10 5 B16F10Luc+ tumor cells were implanted in the mouse ears for 2 ( a – e ) or 4 ( f – j ) weeks. Different parameters were measured on peritumoral and intratumoral areas. ( a , f ) The lymphatic vessel density (LVD). ( b , g ) The number of vessel sections per mm 2 of specific tissue area. ( c , h ) The number of vessel sections smaller than a specific size (<2 × 10 −2 mm 2 or <1 × 10 −2 mm 2 for peritumoral or intratumoral, respectively). ( d , i ) The number of vessel sections larger than a specific size (>2 × 10 −2 mm 2 or >1 × 10 −2 mm 2 for peritumoral or intratumoral, respectively). ( e , j ) The normalized number of vessel sections at a distance of 0.45 mm or 0.3 mm from the ear border after 2 weeks or 4 weeks of sponge insertion. Results are expressed as mean ± SEM, and Wilcoxon-Mann-Whitney significance test was used to compare the mean parameter values, * p < 0.05; ** p < 0.01; *** p < 0.001, n = 12 sponges per group (6 mice/group).

Article Snippet: Murine B16F10Luc+ melanoma cells expressing luciferase (Luc+) (Caliper Life Sciences, Hopkinton, MA) were used in this study.

Techniques: MANN-WHITNEY

Journal: Scientific Reports

Article Title: Modeling pre-metastatic lymphvascular niche in the mouse ear sponge assay

doi: 10.1038/srep41494

Figure Lengend Snippet: Draining sentinel LNs were resected from mice with either control ear sponges or sponges populated with B16F10Luc+ tumor cells. ( a ) Representative sections, belonging to the central LN region, are shown for control SLN and pre-metastatic SLN after 2 weeks of sponge insertion. ( b ) 3D reconstruction of the representative control and pre-metastatic SLN shown in ( a ), with lymphatic vessels in green and tissue border in blue. ( c ) Representative sections of control SLN and metastatic SLN, belonging to the central LN region, after 4 weeks of sponge implantation. ( d ) 3D reconstruction of the representative control and metastatic SLN shown in ( c ), with lymphatic vessels in green, tumor mass in red and tissue border in blue. Right panels in ( a ) and ( c ) represent higher magnification pictures of areas delineated by the square. Immunostained lymphatic vessels (LYVE-1 positive) appear in blue and tumor cells (tyrosinase positive) in pink. Black arrows indicate representative lymphatic vessels and the black dotted line delineate the tumor area. Scale bar represents 250 μm. For 3D reconstructions of SLN, around 40 sections were stacked, with a final z-stack thickness of ∼ 200 μm.

Article Snippet: Murine B16F10Luc+ melanoma cells expressing luciferase (Luc+) (Caliper Life Sciences, Hopkinton, MA) were used in this study.

Techniques: Control