phospho histone h2a x ser139 d7t2v mouse mab  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: The Interstitial Gland as a Source of Pro- or Anti-Senescent Cells during Chinchilla Rabbit Ovarian Aging

doi: 10.3390/ijms25189906

Figure Lengend Snippet: Antibodies and epitope retrieval used for immunolocalization.

Article Snippet: Mouse anti-p-Histone H2A.X (sc-517348, Santa Cruz Biotechnology, Dallas, TX, USA) , 1:25 (IH) 1:200 (WB) , 1X Diva Decloaker (Biocare Medical, Pike Lane, CA, USA).

Techniques: Immunohistochemistry, Western Blot, Plasmid Preparation

Journal: Scientific Reports

Article Title: Additive genotoxic effects in cord blood cells upon indirect exposure to chemotherapeutic compounds crossing an in vitro placental barrier

doi: 10.1038/s41598-024-62250-5

Figure Lengend Snippet: Characterization of a chemotherapy-induced bystander effect by an exposed BeWo bilayer to human cord blood cells. Cord blood mononuclear cells were exposed to either ‘conditioned media’ obtained from BeWo barriers that were treated either with cyclophosphamide (CP, 128 µM) or epirubicin (EPI, 16.6 µM), referred to as ‘indirect exposure’, or to culture medium spiked with the concentrations of CP (38.4 µM, i.e. 30% of 128 µM) or EPI (231 nM, i.e. 1% of 16.6 µM) that were shown to cross the BeWo barrier, named as ‘direct exposure’. Vehicle-treated condition consisted of culture medium supplemented with water at the equivalent volume of the chemotherapeutic compounds. After a 24 h-exposure, DNA damage was measured in the exposed cord blood mononuclear cells. All the values were normalized to the vehicle exposure (reference orange line) and expressed as a fold change. ( A ) The fraction of cells with double strand breaks (γ-H2AX) was determined using FACS, showing that the γ-H2AX-positive fraction increased in all conditions compared to the vehicle-treated reference (reference orange line). The difference was only statistically significant for the ‘indirect exposure’ conditions. Furthermore, for EPI, the increase associated with the ‘indirect exposure’ was statistically significantly higher than seen with the ‘direct exposure’. ( B ) Measuring of nuclear ROS levels, expressed as the mean green intensity that represents the signal emitted by the fluorogenic probe upon oxidation and consequent binding to DNA. Direct exposure to both CP and EPI did not change the levels of ROS in the nuclei of cord blood mononuclear cells compared to the vehicle. In contrast, indirect exposure to CP or EPI increased the levels more than 50% (> 1.5-fold) compared to the vehicle. Plots represent the mean ± SD of 3 independent experiments with 3-technical replicates per experiment. * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by the unpaired t-test, both relative to vehicle and direct exposure. Orange asterisks refer to significant differences when comparing to the vehicle condition. Black asterisks indicate significant differences between direct and indirect exposure conditions.

Article Snippet: Cells were washed with the eBioscience™ Permeabilization Concentrate and Diluent following the manufacturer instructions (Invitrogen) and stained with 1) (1:20) mouse anti-H2A.X Phospho (Ser139) AF488 (AB_2248011); 2) (1:20) rabbit anti-Caspase-3 , AF 647, Clone: C92-605 (BD) and 3) (1:20) mouse anti-Ki-67 APC-eFluor 780, Clone: 20Raj1 (Invitrogen™) for 1 h at room temperature in the darkness.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Additive genotoxic effects in cord blood cells upon indirect exposure to chemotherapeutic compounds crossing an in vitro placental barrier

doi: 10.1038/s41598-024-62250-5

Figure Lengend Snippet: Characterization of a chemotherapy-induced bystander effect by an exposed BeWo bilayer to human cord blood cells. Cord blood mononuclear cells were exposed to either ‘conditioned media’ obtained from BeWo barriers that were treated either with cyclophosphamide (CP, 128 µM) or epirubicin (EPI, 16.6 µM), referred to as ‘indirect exposure’, or to culture medium spiked with the concentrations of CP (38.4 µM, i.e. 30% of 128 µM) or EPI (231 nM, i.e. 1% of 16.6 µM) that were shown to cross the BeWo barrier, named as ‘direct exposure’. Vehicle-treated condition consisted of culture medium supplemented with water at the equivalent volume of the chemotherapeutic compounds. After a 24 h-exposure, DNA damage was measured in the exposed cord blood mononuclear cells. All the values were normalized to the vehicle exposure (reference orange line) and expressed as a fold change. ( A ) The fraction of cells with double strand breaks (γ-H2AX) was determined using FACS, showing that the γ-H2AX-positive fraction increased in all conditions compared to the vehicle-treated reference (reference orange line). The difference was only statistically significant for the ‘indirect exposure’ conditions. Furthermore, for EPI, the increase associated with the ‘indirect exposure’ was statistically significantly higher than seen with the ‘direct exposure’. ( B ) Measuring of nuclear ROS levels, expressed as the mean green intensity that represents the signal emitted by the fluorogenic probe upon oxidation and consequent binding to DNA. Direct exposure to both CP and EPI did not change the levels of ROS in the nuclei of cord blood mononuclear cells compared to the vehicle. In contrast, indirect exposure to CP or EPI increased the levels more than 50% (> 1.5-fold) compared to the vehicle. Plots represent the mean ± SD of 3 independent experiments with 3-technical replicates per experiment. * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by the unpaired t-test, both relative to vehicle and direct exposure. Orange asterisks refer to significant differences when comparing to the vehicle condition. Black asterisks indicate significant differences between direct and indirect exposure conditions.

Article Snippet: Cells were washed with the eBioscience™ Permeabilization Concentrate and Diluent following the manufacturer instructions (Invitrogen) and stained with 1) (1:20) mouse anti-H2A.X Phospho (Ser139) AF488 (AB_2248011); 2) (1:20) rabbit anti-Caspase-3 , AF 647, Clone: C92-605 (BD) and 3) (1:20) mouse anti-Ki-67 APC-eFluor 780, Clone: 20Raj1 (Invitrogen™) for 1 h at room temperature in the darkness.

Techniques: Binding Assay