Cellinsight cx7 high content screening platform

Cells on a chamber slide were incubated with anti-O4 antibodies (1:200; #MAB345, Sigma-Aldrich) for 5 min and fixed in 4% paraformaldehyde solution for 15 min at 37 °C. Following permeabilization with PBS containing 0.2% Triton X-100 and 3% goat serum for 1 h, cells were incubated with Cy3-conjugated anti-GFAP (1:200; C-9205, Sigma-Aldrich) and anti-PU.1 antibodies (1:500; 2258S, Cell Signalling) at 4 °C. The next day, cells were stained using Alexa Fluor 488-conjugated anti-mouse IgM antibodies (1:500; A21042, Invitrogen), Alexa Fluor 647-conjugated anti-rabbit antibodies (1:500; A27040, Invitrogen) and Hoechst 33342 (1 mg/mL; Invitrogen). Percentage of cells positive for GFAP, O4 or IBA1 staining in astrocyte culture was quantified using a CellInsight CX7 High Content Screening Platform (ThermoFisher). All conditions were assessed in triplicate.

CellEvent Caspase 3/7 Detection Reagent is cell-permeant reagent and consists of the peptide (DEVD) conjugated to a nucleic acid-binding dye. Upon cleavage of the DEVD recognition sequence by caspases 3/7, the dye is released to bind cellular DNA increasing its fluorescent signal. For detection, H9c2 cells were plated at 70% confluency in 96-well plates (non-tissue culture coated) and treated with FeSO₄ (100 µM, 24 h) with or without co-treatment with antioxidants NAC (500 nM) and SkQ1 (20 nM). At the treatment endpoint, cells were incubated with Caspase 3/7 Detection Reagent (10 µM) and Hoechst (100 nM) for 30 min at 37 °C protected from light. Afterward, images were obtained and analyzed to characterize cells with activated Caspase 3/7 pathways through the CellInsight CX7 High Content Screening platform (Thermo Fisher, Waltham, MA, USA), Absorption/Emission: 511/533 nm.

The cells were seeded in a 48-well plate at a density of 8,000 cells per well and cultured under the same conditions as mentioned previously. After glutamate treatment for 12 h, the cells were subjected to staining with MitoTracker® Orange CMTMRos (25 nM) and incubated at 37 °C for 30 min. Subsequently, the cells were washed three times with PBS and fixed using 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). For nucleus staining, the cells were stained with DAPI (10 μg/ml) for 10 min at RT and washed with PBS. The fluorescence intensity was quantified and visualized using the Thermo Scientific™ CellInsight CX7 high-content screening platform.

Cells were fixed in 4% paraformaldehyde (PFA, Sigma) in PBS for 20 min at 4 °C followed by a 1 h room temperature incubation in blocking solution of 5% donkey serum (Biosera) in 0.3% Triton-X-100 (Sigma) in PBS (0.3% PBST). Primary antibodies, used at an assay dependent concentration (see ‘Antibody concentration’), were diluted in blocking solution and incubated with cells overnight at 4 °C. Following removal of primary antibody solution and 3 PBS washes, cells were incubated in the dark for 2 h at room temperature with appropriate Alexa Fluor secondary antibodies (Thermo Fisher Scientific) diluted 1:500 with blocking solution. After an additional 2 PBS washes cells were counterstained with DAPI nucleic acid stain (Thermo Fisher Scientific), diluted 1:1000 with PBS, for 5 mins at room temperature and following a final PBS wash, mounted using Dako Fluorescence Mounting Medium (Agilent) and glass coverslips. Imaging was with either the LSM710 confocal microscope (Zeiss) using Zen 2012 SP2 (black) v11.02.190 (Zeiss), LAS X for DMI6000B inverted microscope (Leica) or Cellinsight Cx7 High-Content Screening Platform (Thermo Fisher Scientific) with HCS Studio Cell Analysis software v6.6.0 (Thermo Fisher Scientific) used for quantification.

C-TACs were enriched and harvested from Peripheral Blood Mononuclear Cells (PBMCs) as described previously [10 (link)]. Briefly, PBMCs were treated with epigenetically activating media for up to 100 h at 37 °C under 5% CO₂, 4% O₂. This process induces cell death in normal (non-malignant) cells with functional apoptotic machinery while simultaneously conferring survival privilege on apoptosis-resistant cells of tumorigenic origin, i.e. circulating tumor-associated cells (C-TACs) and their heterotypic clusters (C-ETACs: circulating ensembles of tumor-associated cells). C-TACs (EpCAM + , PanCK + , CD45 ±) include CTCs (EpCAM + , PanCK + , CD45-) as well as other cell types such as tumor-associated macrophages (TAMs) and tumor-associated fibroblasts (TAFs). Supplementary Table S1 provides the C-TAC yields in various cancer types and based on treatment status. Enriched and harvested C-TACs were identified by immunocytochemistry (ICC) profiling for EpCAM, Pan-CK and CD45, as well as Organ and Subtype-Specific (OSS) markers to verify cancer type (Supplementary Figure S1, Supplementary Figure S2, Supplementary Table S2). Fluorescence imaging was performed on Cell Insight CX7 High-Content Screening Platform (ThermoFisher Scientific, USA).