Facsuite

For flow cytometry-based evaluation of the tumor cell frequency in the bone marrow and spleen, 1 × 10⁵–1 × 10⁶ cells in 100 µL were stained with anti-human CD45-FITC (clone 2D1) and anti-human CD19-PE (clone 4G7; both Becton Dickinson, Heidelberg, Germany) at 4 °C for 15 min and washed twice (180× g, 8 min) in cold PBS before measuring using FACSCalibur or FACSVerse and CellQuest Pro or FACSuite software tools (all Beckton Dickinson), respectively. For determination of the peripheral blood blast frequencies, 20–60 µL of blood were collected from the tail vein and stained as mentioned above. Subsequently, erythrocytes were lysed during 15 min of incubation with 2 mL erythrocyte lysis buffer. Cells were washed twice in 2 mL PBS and analyzed by flow cytometry.

Cells were seeded at 1 × 10⁴ per well in a 6-well plate and incubated for 24 h. Cells were detached using 0.5% trypsin (Thermo Fisher Scientific), suspended in 2 mL of PBS then collected in a conical centrifuge tube. Samples were centrifuged at 220× g for 3 min, then suspended in 1 mL PBS + 0.3% BSA (Sigma-Aldrich). All samples were filtered using a 100 µm Corning cell strainer (Thermo Fisher Scientific). Cells were counted using TC20^TM Automated Cell Counter (Bio-Rad Laboratories, Hercules, CA, USA). Cell density was set to 1 × 10⁶, and 25 µL of cell suspension was then incubated with 25 µL FITC pre-conjugated primary antibodies (mouse anti-human integrin β3; 11–0519-42, 1:200; Thermo Fisher Scientific) or isotype control antibodies (mouse IgG kappa isotype control; 11–4714-42, 1:200; Thermo Fisher Scientific) for 30 min at room temperature in U bottom 96 well plates. Samples were centrifuged at 260× g for 2 min. Cells were then washed three times in 100 µL PBS + 0.3% BSA, then resuspended in 200 µL PBS + 0.3% BSA. The samples were transferred into a FACS tube with 200 µL FACS sheath fluid. Cells were analyzed by flow cytometry using a FACSVerse flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). A minimum of 10,000 cells were analyzed per sample. Data were collected and analyzed using FACSuite and FlowJo software packages (Becton, Dickinson and Company).

Apoptosis was analyzed by annexin V/PI staining and subsequent flow cytometry. After inhibitor incubation cells were harvested and washed twice with PBS. Cells were resuspended in binding buffer, stained with annexin V-FITC (Becton Dickinson) and incubated at room temperature protected from light for 15 min. Propidium iodide (0.6 µg/mL) was added just before analysis (FACSCalibur or FACSVerse, Becton Dickinson) and data was analyzed using CellQuest or FACSuite software (both Becton Dickinson).

For patient-derived xenograft (PDX) model generation 2.5 × 10⁶ cells per primary sample were orthotopically xenografted into 8–12 week old male or female NOD scid gamma mice (NOD.Cg-Prkcd^scid Il2rg^tm1Wjl/SzJ, NSG, Charles River Laboratories, Sulzfeld, Germany). Tumor cell engraftment was regularly evaluated by peripheral blood flow cytometry (anti-human CD45-FITC (clone 2D1) and anti-human CD19-PE (clone 4G7)) measured using FACSVerse and FACSuite software, all Becton Dickinson, Heidelberg, Germany). Mice were euthanized by cervical dislocation when a blast frequency of 30% in peripheral blood was achieved and then bone marrow and spleen cells were isolated. Tumor cells were then serially transplanted into the next PDX generation until generation 3. Mice were bred and housed under specific pathogen-free conditions with access to water and standard chow ad libitum. All experiments were carried out in a laboratory setting and no intervention was performed within the animal housing and breeding rooms. Experiments were approved by the review board of the federal state Mecklenburg-Vorpommern, Germany (reference number: LALLF MV/7221.3–1.1-002/15).

Flow cytometry was used for both cell cycle analysis and quantification of infection. For cell cycle analysis, cells were washed with PBS, fixed with ice-cold 70% ethanol, and stained with 0.1% (v/v) Triton X-100, 20 μg/ml propidium iodide (PI) (Sigma, St. Louis, MO, USA) and 100 μg/ml DNase-free RNase (Life Technologies, Grand Island, NY, USA). For the infection assay, cells were disassociated by trypsin and washed with PBS. The infected GFP⁺ cells and uninfected cells were analyzed and quantified by a BD FACSVerse™ flow cytometer (BD Biosciences, San Jose, CA, USA). The FACSuite (BD Biosciences, San Jose, CA, USA) and the FlowJo (Ashland, OR) software were used for data analysis.

EDTA‐anticoagulated fresh bone marrow aspirates from 72 patients of our internal cohort were analyzed. Surface and intracellular antigen detection was performed on fresh bone marrow samples within 2 hours by multicolor flow cytometry. Leukemic stem and progenitor cells (LSC /LPC) were gated according to their CD45/CD34 properties. APC‐Cy7‐labeled anti‐CD45 (mouse monoclonal; clone2D1; #348795), PE‐Cy7‐labeled anti‐CD34 (mouse monoclonal; clone8G12; #348791), PE‐labeled anti‐Tim‐3 (mouse monoclonal; clone7D3; #563422), and isotype matched control antibodies (mouse IgG1 κ APC‐Cy7, #348792; mouse IgG1 κ PE‐Cy7, #348788; mouse IgG1 κ PE,#554680) were purchased from BD Biosciences (BD Biosciences, Franklin Lakes, NJ, USA). For surface antigen staining, 1 × 10⁶ cells were incubated for 30 min with 10 μl of appropriately diluted monoclonal antibody conjugates. The CD45, CD34, and Tim‐3 protein expression of AML specimens from the internal cohort were detected by FACSCantoII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with FACSuite (BD Biosciences) software, and the data were analyzed by FlowJo software (version 10.5.3).

The ROS and lipid ROS levels in the cell lines were measured using flow cytometry. The indicated cell lines were seeded in six‐well plates at a density of 3 × 10⁵ cells per well before the experiment. On the second day, the cells were collected, incubated with PBS containing 25 × 10⁻⁶m DCFH‐DA (35 845, Sigma‒Aldrich) or 5 × 10⁻⁶m C11‐BODIPY 581/591 (RM02821, ABclonal) at 37 °C for 30 min, resuspended in 500 µL of fresh PBS, and analyzed using a flow cytometer (FACSuite, BD Biosciences) equipped with a 488 nm laser for excitation. Measurement of lipid ROS levels in tissues required the production of single‐cell tissue suspensions using collagenase IV (C4‐BIOC, Sigma‒Aldrich) and hyaluronidase (Solarbio, H8030). Single‐cell suspensions were incubated with the Zombie NIR Fixable Viability Kit and 5 × 10⁻⁶m C11‐BODIPY 581/591, as described previously. Dead cells were excluded by setting the gate with the Zombie Viability Dye, and the lipid ROS levels of viable cells were detected. Data were collected from the FITC and APC‐Cy7 channels, and the mean fluorescence was analyzed using FlowJo Version 10. 8 software.