lsm710/810 zen blue confocal microscopy system  (Carl Zeiss)

Journal: Cell Adhesion & Migration

Article Title: Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

doi: 10.1080/19336918.2015.1035504

Figure Lengend Snippet: Haptoglobin expression in the SKOV-3 ovarian cancer cell line and primary culture cells (INCan017 and INCan019). ( A ) RT-PCR assays using specific primers for Hp, showing an amplification product corresponding to 510 bp; ( B ) Western blot ( C ) and confocal microscopy ( C ) analyses using an anti-haptoglobin monoclonal antibody (1:1000, and 1:100, respectively) of cells that were harvested from culture medium or ascitic fluid. The cells in culture medium did not express the protein, whereas these cells, when stimulated with ascitic fluid from an ovarian cancer patient, expressed the haptoglobin protein. Bar scale = 100 μm.

Article Snippet: However, under Hp conditions, the cells exceed 2 or 3 times the size of control cells or of those exposed to ascitic fluid; they were rounded in shape, forming short actin filaments, podosome-like structures, presenting lamellipodia in its membranes, and had plenty of actin points (most likely nucleation centers) and an actin disposition that was very similar to concentric swirls toward the nucleus, with long filaments of actin close to the cell membrane in close contact with the adhesion zone ( ). confirms the results described above by a quantitative analysis via confocal microscopy Zen Blue Edition Software (Carl Zeiss).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Confocal Microscopy

Journal: Cell Adhesion & Migration

Article Title: Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

doi: 10.1080/19336918.2015.1035504

Figure Lengend Snippet: CCR2 (chemokine receptor-2) expression in the ovarian cancer cell line SKOV-3 and primary culture cells (INCan017 and INCan019). ( A ) Total extracts of tumor cells were separated by electrophoresis and transferred to a nitrocellulose membrane that was incubated with the primary antibodies (α-CCR2, No. Cat. 21667, Abcam; α-ERK 1 (K-23), No. Cat. SC-94, Santa Cruz Biotech, as a loading control). The ovarian cancer cells that were harvested from the culture medium did not express the protein; this result was corroborated by confocal microscopy (10 5 cells) using an anti-CCR2 antibody (1:100). ( B ) When the cells were stimulated with ascitic fluid from an ovarian cancer patient, CCR2 protein expression was detected by Western blot and corroborated by confocal microscopy. Bar scale = 100 μm.

Article Snippet: However, under Hp conditions, the cells exceed 2 or 3 times the size of control cells or of those exposed to ascitic fluid; they were rounded in shape, forming short actin filaments, podosome-like structures, presenting lamellipodia in its membranes, and had plenty of actin points (most likely nucleation centers) and an actin disposition that was very similar to concentric swirls toward the nucleus, with long filaments of actin close to the cell membrane in close contact with the adhesion zone ( ). confirms the results described above by a quantitative analysis via confocal microscopy Zen Blue Edition Software (Carl Zeiss).

Techniques: Expressing, Electrophoresis, Membrane, Incubation, Control, Confocal Microscopy, Western Blot

Journal: Cell Adhesion & Migration

Article Title: Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

doi: 10.1080/19336918.2015.1035504

Figure Lengend Snippet: Participation of ascitic fluid in migration of tumor cells. ( A ) “Wound-Healing Assays” were carried out to measure the capacity of the ascitic fluid as an inducer agent of migration using confocal microscopy: Nuclei were stained with DAPI (blue, 1:50) and structures of polymerized actin by rhodamine-phalloidin (red, 1:25). ( B ) A quantitative analysis of fluorescence was performed using the Zen 2011 software (Blue edition, Carl Zeiss) considering an average area of 9 × 105 μm 2 ; the program determines the mean fluorescence intensity of each condition, which is plotted in as a histogram. The results were analyzed corresponding to the average of 3 independent experiments, showing a clear increase of fluorescence in both channels due to the migration of cells after 24 h. ( C ) Morphological changes were analyzed by performing a optical zoom of the cells that were exposed to ascitic fluid (the dashed arrows show the formation of filopodia and extensions of unidirectional membrane protrusions). The nuclear localization in each case is shown with solid arrows. Bar scale = 100 μm

Article Snippet: However, under Hp conditions, the cells exceed 2 or 3 times the size of control cells or of those exposed to ascitic fluid; they were rounded in shape, forming short actin filaments, podosome-like structures, presenting lamellipodia in its membranes, and had plenty of actin points (most likely nucleation centers) and an actin disposition that was very similar to concentric swirls toward the nucleus, with long filaments of actin close to the cell membrane in close contact with the adhesion zone ( ). confirms the results described above by a quantitative analysis via confocal microscopy Zen Blue Edition Software (Carl Zeiss).

Techniques: Migration, Confocal Microscopy, Staining, Fluorescence, Software, Membrane

Journal: Cell Adhesion & Migration

Article Title: Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

doi: 10.1080/19336918.2015.1035504

Figure Lengend Snippet: Time-dependent expression of CCR2 in the cells that were exposed to medium, ascitic fluid, and haptoglobin induces changes in actin cytoskeleton. ( A ) Total extracts of the 3 cell types were processed for protein gel blot assays to analyze the expression level of the CCR2 receptor for 0, 24 and 48 h of exposure in the medium and ascitic fluid. ( B ) The same kinetics were analyzed by confocal microscopy with a 40 × objective using an LMS 700 confocal microscope; nuclei were stained with DAPI (blue, 1:50), actin structures with rhodamine-phalloidin (red, 1:25), and CCR2 with a primary anti-CCR2 antibody (1:1000 for western blot and 1:100 for confocal microscopy) and a secondary-FITC conjugated antibody (green). The merged zones are shown in yellow. The cells were stimulated with Hp (purified human cancer-free serum). Bar scale = 100 μm. ( C ) From the cells that were exposed to medium, ascitic fluid or Hp, randomly selected fields to which an optical zoom (crop) was performed were subjected to an ortho analysis to obtain the zx, zy and zz axes from each image. Bar scale = 100 μm. ( D-G ) A quantitative analysis was performed using the software Zen 2011 (Blue edition, Carl Zeiss). A Shapiro-Wilk analysis was performed to evaluate the data distribution. To determine the differences among the conditions (culture medium, ascitic fluid, and Hp), a Mann-Whitney analysis for non-parametric data was applied. The analyses were performed for at least 30 cells under each condition ( P < 0.001).

Article Snippet: However, under Hp conditions, the cells exceed 2 or 3 times the size of control cells or of those exposed to ascitic fluid; they were rounded in shape, forming short actin filaments, podosome-like structures, presenting lamellipodia in its membranes, and had plenty of actin points (most likely nucleation centers) and an actin disposition that was very similar to concentric swirls toward the nucleus, with long filaments of actin close to the cell membrane in close contact with the adhesion zone ( ). confirms the results described above by a quantitative analysis via confocal microscopy Zen Blue Edition Software (Carl Zeiss).

Techniques: Expressing, Western Blot, Confocal Microscopy, Microscopy, Staining, Purification, Software, MANN-WHITNEY

Journal: Cell Adhesion & Migration

Article Title: Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

doi: 10.1080/19336918.2015.1035504

Figure Lengend Snippet: Monocyte chemoattractant protein 1 (MCP1) and haptoglobin (Hp) are affected in their capacity to migrate by CCR2 antagonist (RS1028959 exposition). Wound-healing assays were performed on SKOV-3 cells for 0, 12, 24 and 48 h at the indicated doses of Hp (250 μg) and monocyte chemoattractant protein 1 (MCP1, 250 ng). In addition, the effect of pretreatment with the chemokine (C-C motif) receptor 2 (CCR2) synthetic antagonist RS102895 (5 μM) on the capacity of SKOV-3 cells to migrate toward MCP1 (250 ng/ml) and Hp (0.250 mg/ml) is shown. As a negative control, bovine serum albumin (BSA) (1 mg/ml) was used. Chemotaxis was measured by confocal microscopy; nuclei (blue) were stained with DAPI (1:50). ( B ) Quantitative analysis of migration by the software Zen 2011 (Blue edition, Carl Zeiss) considering an average area of 9 × 10 5 μm 2 . This program determines the mean fluorescence intensity of each condition, which is plotted as a histogram. The results correspond to the average of 3 independent experiments. Bar scale = 100 μm.

Article Snippet: However, under Hp conditions, the cells exceed 2 or 3 times the size of control cells or of those exposed to ascitic fluid; they were rounded in shape, forming short actin filaments, podosome-like structures, presenting lamellipodia in its membranes, and had plenty of actin points (most likely nucleation centers) and an actin disposition that was very similar to concentric swirls toward the nucleus, with long filaments of actin close to the cell membrane in close contact with the adhesion zone ( ). confirms the results described above by a quantitative analysis via confocal microscopy Zen Blue Edition Software (Carl Zeiss).

Techniques: Negative Control, Chemotaxis Assay, Confocal Microscopy, Staining, Migration, Software, Fluorescence