non reactive astrocytes ditnc1 atcc  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: A Pro-Inflammatory Stimulus versus Extensive Passaging of DITNC1 Astrocyte Cultures as Models to Study Astrogliosis

doi: 10.3390/ijms25179454

Figure Lengend Snippet: DITNC1 astrocytes with extensive passaging but not low passages respond to neuronal Thy-1 without inflammatory stimuli. ( A ) Phase contrast microphotographs of low passage (LP), high passage (HP), DITNC1(ATCC), and DITNC1(CH) astrocytes grown for 48 h. Magnification = 200×. ( B ) Immunofluorescence microphotographs obtained with the confocal microscope of LP, HP DITNC1(ATCC), and DITNC1(CH) astrocytes grown for 48 h and stained with DAPI (nuclei, blue) and rhodamine-labelled phalloidin (F-actin, red). Cells clustered leaving free spaces in the plate with a characteristic oval shape (white arrow). Scale bar = 20 µm. ( C ) LP, HP DITNC1(ATCC), and DITNC1(CH) astrocyte lysates were immunoblotted with antibodies against C3d or S100β and β-actin as a loading control. Densitometric and statistical analyses are shown below each band as the mean ± s.e.m. ( D ) LP, middle passage (MP), HP DITNC1(ATCC), and DITNC1(CH) astrocyte lysates were immunoblotted with antibodies against β 3 Integrin and HSP90 as a loading control. This graph shows the averaged β 3 Integrin band intensity normalized to HSP90, the values expressed as ratios, and the fold-change of the mean LP value. ( E ) Insert: A histogram obtained by flow cytometry to evaluate β 3 Integrin levels on the surface of astrocytes. The three cell populations correspond to the LP (blue), HP (orange), and CH (purple) cells, and the isotype control is shown (green). Graph: median fluorescence intensity (MFI) determined by flow cytometry, shown in the histogram (insert). Bars in the graph indicate the values obtained from LP (white), HP (dark gray), and CH (black) cells. ( F ) The migration assay of LP, HP DITNC1(ATCC), and DITNC1(CH) astrocytes was performed as described in Materials and methods. All the cell lines were stimulated with Thy-1-Fc conjugated to Protein A [Thy-1-Fc/Protein-A (4 µg/0.4 µg per well)] for 2 h or with TRAIL-R2-Fc as a negative control. The graph shows values normalized to the average of LP DITNC1(ATCC) astrocytes under the control condition. Values in ( D – F ) correspond to the mean ± s.e.m (n = 3). Significant differences are indicated. * p < 0.05 compared to LP values [or LP/TRAIL-R2 in (F)]. # p < 0.05 compared to each respective control treated with TRAIL-R2. R.U = Relative Units.

Article Snippet: Thus, ATP and polyphosphates are likely critical factors contributing to the observed neurotoxicity in the conditioned medium from DITNC1 reactive astrocytes; however, further investigations are required to identify the toxic factors present in the ACM of LP DITNC1(ATCC) treated with TNF and DITNC1(CH) cells.

Techniques: Passaging, Immunofluorescence, Microscopy, Staining, Control, Flow Cytometry, Fluorescence, Migration, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: A Pro-Inflammatory Stimulus versus Extensive Passaging of DITNC1 Astrocyte Cultures as Models to Study Astrogliosis

doi: 10.3390/ijms25179454

Figure Lengend Snippet: Cells with a low number of passages behave as non-reactive astrocytes and switch to reactive astrocytes when treated with TNF. ( A ) LP DITNC1(ATCC) astrocytes treated with TNF (10 ng/mL) for 48 h (or the indicated periods) or left untreated were subjected to immunoblots, immunofluorescence, cell adhesion, migration, and proliferation assays. ( B ) Immunoblot analysis of GFAP at 24, 48, and 72 h. β-Actin was the loading control. ( C ) Immunoblot analysis of whole LP DITNC1(ATCC) ± TNF (10 ng/mL, 48 h) cell lysates using anti-β 3 Integrin and anti-Cx43 antibodies. β-Actin was used as a loading control. Values below the blots in ( B , C ) are the means ± s.e.m. of the ratio between the densitometric value of the first antibody signal and that of the respective β-Actin value (n = 3). ( D ) Representative images of low passage (LP) ± TNF pretreatment; Vimentin or Cx43 (green), F-actin (rhodamine-conjugated phalloidin in red), and nuclei (DAPI in blue) were visualized (scale bar = 50 µm). White arrows indicate Cx43 localized at the cell border. ( E ) Representative images of low passage (LP) ± TNF pretreatment (outlined rectangular area) and DITNC1(CH) astrocytes. Cells were treated with TRAIL-R2-Fc or stimulated with Thy-1-Fc, as indicated. FAs, F-actin, and nuclei were, respectively, visualized with vinculin staining (green), rhodamine-conjugated phalloidin (red), and DAPI (blue) (scale bar = 20 µm). ( F ) FA quantification. LP DITNC1(ATCC) cells were evaluated under untreated conditions (Control) or with TNF (10 ng/mL, 48 h) (LP + TNF), and DITNC1(CH) cells were stimulated with Thy-1 or treated with the negative control TRAIL-R2. The values in the graph are the means ± s.e.m. of >30 cells monitored per condition in each experiment (n = 3). # p < 0.05 compared to each respective control treated with TRAIL-R2. ( G , H ) Migration assay using LP DITNC1(ATCC) cells ( G ) or DITNC1(CH) cells ( H ) treated with TNF (+TNF) (10 ng/mL, 48 h) or left untreated (-TNF). The transwell assay was performed by adding 10 5 cells in a serum-free medium containing Thy-1-Fc or TRAIL-R2-Fc to the top chamber for 2 h. Astrocytes that migrated to the bottom side through the insert pores were visualized by crystal violet staining and then counted. The graphs show the values normalized to the average of the control condition (LP + TRAIL-R2). At least six fields were counted per condition (n = 3) (mean ± s.e.m.). * p < 0.05; ** p < 0.01. ( I ) Cell proliferation was evaluated using the MTS assay in LP DITNC1(ATCC) cells treated with TNF (LP + TNF) or without TNF (LP) and in DITNC1(CH) cells after incubating them for 24, 48, and 72 h. The values in the graph indicate the optical density (O.D.) at 490 nm measured at the indicated time points (n = 3) (mean ± s.e.m.). *** p < 0.001 compared to LP DITNC1(ATCC) cells not treated with TNF at 72 h.

Article Snippet: Thus, ATP and polyphosphates are likely critical factors contributing to the observed neurotoxicity in the conditioned medium from DITNC1 reactive astrocytes; however, further investigations are required to identify the toxic factors present in the ACM of LP DITNC1(ATCC) treated with TNF and DITNC1(CH) cells.

Techniques: Western Blot, Immunofluorescence, Migration, Control, Staining, Negative Control, Transwell Assay, MTS Assay

Journal: International Journal of Molecular Sciences

Article Title: A Pro-Inflammatory Stimulus versus Extensive Passaging of DITNC1 Astrocyte Cultures as Models to Study Astrogliosis

doi: 10.3390/ijms25179454

Figure Lengend Snippet: DITNC1 cells that undergo extensive passaging lack senescence-associated traits. ( A ) SA-β-galactosidase activity of cells treated with TNF or H 2 O 2 . Representative images of LP DITNC1(ATCC) (LP), HP DITNC1(ATCC) (HP), and DITNC1(CH) cells treated with 10 ng/mL of TNF or 10 µM H 2 O 2 for 48 h. SiHa cells treated with 10 µM H 2 O 2 for 48 h are also shown as a control for SA-β-galactosidase-positive cells. After incubation, SA-β-galactosidase activity was determined as described in Materials and methods. Magnification = 100×. Digital magnification= 3×. ( B ) Cell cycle assessment of TNF-treated cells. LP DITNC1(ATCC) cells treated with TNF (LP + TNF) or without TNF (LP), HP DITNC1(ATCC) cells treated with TNF (HP + TNF) or without TNF, and (HP) DITNC1(CH) cells treated with TNF (CH + TNF) or without TNF (CH). All TNF treatments were for 48 h. Cells were fixed and permeabilized with methanol, treated with RNAse, and stained with propidium iodide to determine the G0/G1 (black bars), S (light grey bars), and G2/M (dark grey bars) cell cycle stages by cell cytometry. The values in the graph are the means ± s.e.m. of three independent experiments.

Article Snippet: Thus, ATP and polyphosphates are likely critical factors contributing to the observed neurotoxicity in the conditioned medium from DITNC1 reactive astrocytes; however, further investigations are required to identify the toxic factors present in the ACM of LP DITNC1(ATCC) treated with TNF and DITNC1(CH) cells.

Techniques: Passaging, Activity Assay, Control, Incubation, Staining, Cytometry

Journal: International Journal of Molecular Sciences

Article Title: A Pro-Inflammatory Stimulus versus Extensive Passaging of DITNC1 Astrocyte Cultures as Models to Study Astrogliosis

doi: 10.3390/ijms25179454

Figure Lengend Snippet: DITNC1 astrocytes with multiple passages inhibit neurite outgrowth and promote neuronal death. ( A ) 1. CAD cells (10,000 cells/cm 2 ) labeled with Cell Tracker Green CMFDA (10 μM) were seeded onto a plate or co-cultured on top of a fixed monolayer of astrocytes [LP DITNC1(ATCC) cells with TNF (LP + TNF) or without TNF (LP), or DITNC1(CH) cells]. The extension of neuronal processes was induced by serum depletion and the addition of sodium selenite 50 ng/mL for 24 h. 2. Astrocyte-conditioned medium (ACM) was obtained from LP DITNC1(ATCC) cells with TNF (LP + TNF) or without TNF (LP) and DITNC1(CH) cells. Differentiated CAD cells were incubated with ACM for 24 h. ( B ) Representative microphotographs of fluorescent CAD cells (green) grown on a plate or over LP DITNC1(ATCC) cells treated with TNF (LP + TNF) or without TNF (LP) or over DITNC1(CH) astrocytes (bright field), obtained with a Cytation 3 instrument (BioTek, Santa Clara, CA, USA). White arrows indicate extended neurites. Magnification = 200×. ( C ) Quantification of neurite length (μm). For each quantification, the neurites of at least 50 cells were measured per condition using NeuronJ. The values in the graph are the mean ± s.e.m. (n = 3). Significant differences are indicated. ** p < 0.01, compared to control values on plates; # p < 0.05, compared to the value of LP + TNF. ( D ) Differentiated CAD cells were incubated with ACM for 24 h, and quantification of cell viability was then performed with propidium iodide (PI) staining. The values in the graph are the mean ± s.e.m. (n = 3). Significant differences are indicated by * p < 0.05 compared to LP control values.

Article Snippet: Thus, ATP and polyphosphates are likely critical factors contributing to the observed neurotoxicity in the conditioned medium from DITNC1 reactive astrocytes; however, further investigations are required to identify the toxic factors present in the ACM of LP DITNC1(ATCC) treated with TNF and DITNC1(CH) cells.

Techniques: Labeling, Cell Culture, Serum Depletion, Incubation, Control, Staining