Cellroxgreen kit

To quantify biofilm-associated leukocytes in the soft tissue surrounding the infected knee, homogenates were filtered (35 μm; BD Falcon, BD Biosciences) and RBCs eliminated using RBC Lysis Buffer (Cat #420301, BioLegend). Cells were washed and incubated with TruStain FcX (Cat #101320, BioLegend) and stained with Live/Dead Fixable Blue Dead Cell Stain (Cat #L23105, Invitrogen), CD45-PacBlue (Cat #103126; RRID:AB_493535), Ly6G-PE (Cat #127608; RRID:AB_1186099), F4/80-PE-Cy7 (Cat #123114; RRID:AB_893478), and CD11b-FITC (Cat #101206; RRID:AB_312789) (all from BioLegend), and Ly6C-PerCP-Cy5.5 (Cat #560525, BD Pharmingen). ROS production was assessed using a CellROX Green kit (Cat #C10492, ThermoFisher) according to the manufacturer’s instructions. For individual samples, 10,000–100,000 events were analyzed using BD FACSDiva software (RRID:SCR_001456) with cell populations expressed as the percentage of total viable CD45⁺ leukocytes as previously described [15 (link)].

ROS were analyzed using Cell ROX Green kit (Life technologies, New York, USA) according to manufacturer’s instructions. For positive control, cells were treated with Tert-butyl hydrogen peroxide (TBHP) (working concentration 200 µM). Briefly, after exposure or sham exposure, 1 million cells in 500 µl were taken for ROS measurement and 2 µl of 2.5 mM Cell ROX solution was added directly to the cells in RPMI and incubated for 45 min in CO₂ incubator with closed caps. After 20 min of incubation antibodies against surface markers were added, specifically, CD45-V450 conjugate (BD biosciences, San Jose, California, USA), CD34-APC conjugate (Myltenyi Biotec, Bergisch Gladbach, Germany) specific for HSPC, CD38-PeCy7 (BD biosciences) conjugate to distinguish population that is enriched for either HSC (CD34+ 38−) or progenitors (CD34+ 38+) and 3 µl of 7-AAD for dead cells staining. Samples were then incubated for another 25 minutes in CO₂ incubator and subsequently analyzed by flow cytometer (BD FACS Canto II). Data were analyzed via BD FACS Diva software. Compensation matrix was created by the compensation wizard in the FACS Diva software after acquisition of single color stained samples and unstained control.

For analysis of ROS, the samples were processed essentially, as previously described using Cell ROX Green kit (Life technologies, Grand Island, NY, USA) [46 (link)]. Briefly, AML PGF positive and negative samples were simultaneously thawed, incubated 3 h, and one million cells were taken for ROS measurement. Subsequently, Cell ROX solution and antibodies against surface markers were added, specifically CD45-V450 (BD biosciences) for lymphocyte cells, CD34-APC (Myltenyi Biotec, Bergisch Gladbach, Germany) for HSPCs, and 7-AAD (BD biosciences, San Jose, CA, USA) for dead cells. After 45 min. of incubation, the samples were analyzed by ImageStreamX-100 (Amnis). The data were analyzed via Inspire software. For positive control, cells were treated with Tert-butyl hydrogen peroxide (200 µM).