Live dead fixable green dead cell stain kit

To identify proliferating cells, mast cell lines were stained with 5 µM of Cell Trace Violet (Thermo Fischer Scientific) in PBS following manufacturer’s guidelines. After washing, 5x10⁵ cells were plated in 6 well plates (2 mL/well) with or without the indicated concentrations of inhibitors and cultured for 72 h (HMC-1.1 and HMC-1.2) or 7 days (LAD2) in culture media. To determine viability, cells were collected and stained with a LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Thermo Fischer Scientific) in PBS for 10 min on ice. Data acquisition was performed on a LSRFortessa™ and analyzed using FlowJo software. After gating out dead cells (LIVE/DEAD™ stained), generations of proliferating cells were recognized by diminishing fluorescence intensity. Cultures of BMMC were stained with 5 µM of Cell Trace Violet (CTV), plated in 6-well plates (5x10⁵ cells in 2 mL) and after 6 days, proliferation and cell viability were analyzed as described above. Cultures of hMC differentiated for 7-9 weeks were plated in 24-well plates (2.5x10⁵ cells in 1 mL) in growth media with or without the indicated concentrations of inhibitors, and cell viability was analyzed after 5 days as described above.

In vitro assays were performed using either freshly isolated or thawed thymocytes. Thawed thymocytes were treated with DNase I (STEMCELL, Vancouver, Canada) for 15 min to prevent cell clumping. Dead cells were separated from live cells by staining with LIVE/DEAD™ Fixable Green Dead Cell Stain Kit, for 488 nm excitation (ThermoFisher) for 30 min and sorting with BD FACSJazz™ cell sorter. Live cells were collected in FBS and were used for cell culture.

PARP1 dicer-substrate siRNA (dsiRNA) and scrambled non-targeting negative control dsiRNA (Integrated DNA Technologies) were introduced into 1 × 10⁶ cells in the logarithmic growth phase using nucleofection (Lonza 4D-Nucleofector X Unit: AAF-1003X, 16-well nucleocuvette strips). Cells were nucleofected according to the manufacturer’s guidelines; SF solution and program DN-100 were used for the nucleofection of OCI-AML2 cell line, and SF solution and program EH-100 was used for the nucleofection of OCI-AML3 cell line. Nucleofected cells were validated for gene silencing using RT-qPCR and Western blot. Furthermore, nucleofected cells were treated with escalating doses of ara-C and Idarubicin in a 17:1 ratio for 24 h and assessed for cell viability using the LIVE/DEAD fixable green dead cell stain kit (Thermo Fisher Scientific) and flow cytometry according to the instructions of the manufacturer.

Cells were seeded onto pNFSs and incubated in a humidified 5% CO₂/95% air incubator at 37 °C. After 3 days, pNFSs were stacked in four layers and incubated for 3 days. During the latter 3-day incubation period, designated samples of 4-layer pNFSs were separated into individual layers on each day, and cells in each layer were analyzed for viability using a LIVE/DEAD Fixable Green Dead Cell Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA). The fluorescence of dead (green) cells was analyzed using a C1-Plus laser-scanning TE2000E confocal microscope (Nikon, Tokyo, Japan).

OCI-AML2 (passage range of p7 to p9) and OCI-AML3 (passage range of p2 to p4) cells were seeded in 6-well plates at a density of 4 x 10⁵/2 mL media per well and incubated with escalating doses of ara-C and idarubicin in 17:1 ratio for 24 h. 1 μM olaparib pretreatment was done 24 h prior to ara-C/Idarubicin treatment. Drug-treated cells were centrifugated at 500 xg for 5 min, and the pellet was washed twice with PBS supplemented with 2% FBS. Primary antibodies Biotin anti-mouse/human CD11b antibody (BioLegend) and BUV496 Mouse Anti-Human CD14 antibody (BD Biosciences) were diluted in 2% FBS/PBS to their appropriate concentrations and cells were incubated on ice in the dark with 100 μL of the solution for 25 min. Cells were washed with 2% FBS/PBS, and cells treated with the primary antibody Biotin anti-mouse/human CD11b (BioLegend) were incubated on ice in the dark in 100 μL of 2% FBS/PBS containing the secondary antibody Streptavidin APC-Cy7 (BD Biosciences) for 25 min. All conditions were washed and stained for live and dead cells with the LIVE/DEAD fixable green dead cell stain kit (Thermo Fisher Scientific) according to the instructions of the manufacturer. Cells were transferred to FACS tubes, analyzed by LSR II Fortessa (BD Biosciences), and the results were evaluated with the FlowJo software (version 10.0.8, FlowJo LLC).