H2a x phosphorylation assay kit

The glioma cells were seeded in a 6-well plate at a density of 2 × 10⁵ cells/well and treated with Rc TR extracts (0.75 mg/mL) for 24 h. The cells cultured in the absence of the Rc TR extract were used as the control. The phosphorylated H2A.X- and cleaved PARP-positive cells were measured using Apoptosis, DNA Damage and Cell Proliferation Kit (BD Pharmingen, San Jose, CA, USA, 562253) according to the manufacturer’s protocol. In this experiment we used only two compounds of the mentioned kit—Alexa Fluor^® 647 Mouse Anti-H2A.X (pS139) and PE Mouse Anti-Cleaved PARP (Asp214) Antibodies. All experiments were performed using a FACS Canto II cytometer (Becton–Dickinson, San Jose, CA, USA).
The level of γ-H2A.X was also measured using an H2A.X Phosphorylation Assay Kit (Millipore, Billerica, MA, USA) according to the protocol of the manufacturer. Chemiluminescence detection was performed using attached HRP-substrates using a GloMax-Multi device (Promega, Madison, WI, USA).

The primary H7PX glioma cell line was cultured at a density of 1 × 10⁴ cells per well with vehicle or with the following abietane diterpenes: Deroy, Diroy, Roy and Parv D. They were administered at the IC₅₀ concentration on black 96-well plates with a clear bottom. The level of phosphorylated H2A.X histone was determined using an H2A.X Phosphorylation Assay Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. Briefly, chemiluminescence detection was performed using a GloMax-Multi device (Promega). The mixtures were incubated for 30 min with 35 μM. Bleomycine as a positive control.

The human histone H2AX protein is well conserved in T. adhaerens (TriadG64252, 82% identity). In particular, serine 139 is present both in human and T. adhaerens protein (BLAST [51 (link)]). Thus, we used the H2A.X Phosphorylation Assay Kit (Flow cytometry, Millipore, Catalog # 17–344) to detect the level of phosphorylated (serine 139) histone H2AX. We collected 100 animals immediately after the X-ray exposure for each control and experiment replica (3 biological replicas of the experiment) in ASW. We then removed the ASW, and we dissociated the cells in cold Mg⁺⁺ and Ca⁺⁺ free PBS with 20 mM EDTA by gently pipetting the collected animals soon after adding the PBS-EDTA buffer. The samples were then kept on ice for 5 minutes to ensure complete dissociation of cells. We processed the dissociated cells according to the manufacturer’s specifications, and we quantified the level of phosphorylated histone H2AX using an Attune NxT Flow Cytometer (Invitrogen).

Cell-cycle analyses were performed with a propidium iodide flow cytometry kit (ABCam, 139418, Cambridge, UK) according to the instructions of the manufacturer. A H2AX phosphorylation assay kit (Millipore, 17–344, Vienna, Austria) was used to analyze DNA damage by flow cytometry. At least 10,000 stained cells were analyzed per experimental point in a FACS BD LSR II (Becton Dickinson, Schwechat, Austria) with FACS Diva Software (Becton Dickinson Biosciences, Schwechat, Austria); the flow cytometry gates remained "fixed" in each experimental series according to the controls for each experimental series. All experiments were performed in triplicate.

Cisplatin, oxaliplatin, irinotecan, 5-fluoruracil, gemcitabine and paclitaxel were purchased from Sigma, and used at the concentrations indicated. For knockdown studies, 50K cells were seeded per 6-well plate well, transfected with siRNA the following day, and then fresh media with chemotherapeutic agent (or vehicle control) added one day later. For re-expression studies, 50K cells were seeded per 6-well plate well, and then fresh media with chemotherapeutic agent added one day later. Cell viability was measured by WST-1 assay (Roche) following 72 hrs treatment, and IC₅₀ values determined by fitting a sigmoidal curve (GraphPad Prism). All assays were done in biological triplicate. γH2AX levels (a surrogate for γH2AX foci) were quantified by flow cytometry (> 10,000 cells) using a FITC-conjugated anti-phospho-H2A.X(Ser139) antibody (H2A.X Phosphorylation Assay Kit; EMD Millipore). Apoptosis was measured by flow cytometry (> 10,000 cells) by dual YO-PRO-1 and PI staining (Membrane Permeability/Dead Cell Apoptosis Kit; Invitrogen).

To compare the amount of DNA damage caused by etoposide alone and in combination with other drugs, we measured the levels of phosphorylated H2A.X in HL-60 cells using flow cytometry. We stained the fixed HL-60 cells using the H2A.X phosphorylation assay kit (Merck, Germany) according to manufacturer’s instructions. In short, 5 × 10⁵ HL-60 cells were seeded per well in a 6-well plate and incubated overnight. Cells were treated for 24 hours with IC₂₅ concentration of etoposide alone and in combination with other drugs. Next, cells were harvested and washed with PBS followed by fixation. Cells were then stained with either FITC-conjugated anti-phospho-Histone H2A.X (Ser139) or with the negative control mouse IgG-FITC conjugate for 20 minutes on ice. The amount of H2A.X was then measured using BD Accuri flow cytometer. The data was then analyzed using FlowJo software (v10).