Cyan adp instrument

Flow cytometric analysis of endothelial and cardiomyocyte oxidative stress and IL-36R was performed as previously described (15 (link)). Briefly, hearts were collagenase-digested to obtain single-cell suspensions. Cells were incubated with antibodies: anti-DNA/RNA damage (1:100 dilution, Abcam), anti-IL-36R (1:100 dilution, R & D Systems; Alexa-647 secondary, 1:100 dilution, Biolegend), anti-CD31 (1:100 dilution, Biolegend), anti-cTnT (1:100 dilution, Miltenyi Biotec), and Zombie dye (1:500 dilution, Biolegend), along with appropriate IgG controls. Flow cytometry using a CyAn™ ADP instrument (Beckman Coulter, USA) captured 250,000 events per sample. Data analysis was performed with Summit 4.3 software (Beckman Coulter, USA).

Cells were resuspended in PBS containing 1% BSA and 0.1% sodium azide incubated with optimal concentrations of fluorochrome-conjugated Abs for 30 min at 4°C, washed, and fixed with 1% paraformaldehyde. A PE-conjugated anti-IL-2Rα-chain Ab (CD25, clone OX-39, mouse IgG1), an APC-conjugated anti-CD4 Ab (clone OX-35, mouse IgG2a), a PerCP-conjugated anti-CD8α chain Ab (clone OX-8, mouse IgG1), a FITC-conjugated anti-CD45R Ab (a marker of B cells, clone HIS24, mouse IgG2b), and appropriate isotype controls were purchased from BioLegend (San Diego, CA). An efluor 450-conjugated mAb against Foxp3 (Clone FJK-16s, rat IGg2a) and fixation and permeabilization buffers were purchased from eBioscience (San Diego, CA). Flow cytometry was performed using a CYAN ADP instrument (Beckman Coulter), and the results were analyzed with FlowJo software.

To quantify the level of leukemic engraftment in the orthotopic AML xenograft model, we labeled the PB and BM MNCs with the anti–huCD45-FITC (BD Pharmingen, catalog 555482) antibody following red cell lysis. The cells, which had been previously washed with PBS, were stained for 30 minutes at 20°C. After washing, the samples were analyzed with the Gallios flow cytometer (Becton Dickinson).
Similarly for the FLT3-ITD–transduced primary murine HPCs, the level of engraftment in the BM and spleens of primary recipients was analyzed based on GFP expression.
For apoptosis analysis, harvested cells were stained using the annexin V–PE apoptosis detection kit (BD Pharmingen). Cells were washed with PBS and then resuspended in 100 μl of binding buffer and incubated with PE-conjugated annexin V and 7AAD. The mixture was incubated at room temperature for 15 minutes before flow cytometric analysis with the Cyan ADP instrument (Beckman Coulter).