Flow check pro fluorospheres

EVs were analyzed on a Navios flow cytometer (Beckman Coulter, Brea, CA, USA) as previously described [19 (link),20 (link)]. Briefly, fluorescence settings were tested daily using Flow-Check Pro Fluorospheres (Beckman Coulter, #A63493). Prior to labeling, antibody solutions were centrifuged at 18,000× g for 5–10 min in order to eliminate potential protein aggregates. For labeling with CD34-PE or CD81-PE antibodies (Beckman Coulter, #A07776, and Miltenyi Biotech, #130-118-481, respectively), 15 μL PFP was diluted to 100 μL with 0.1 μm-filtered Cell Wash Buffer (CWB, BD) containing 2% BSA and incubated overnight with 5 μL of the pure antibody, at room temperature in the dark. Samples were then diluted to 500 μL with CWB for FT-FCM analysis. As negative control for antibody labeling, PFP samples were prepared as described above, with CWB supplemented with 0.1% Triton X-100 (AxonLab, Stuttgart, Germany). As reagents-only control, samples were prepared with CWB containing 2% BSA and either CD34-PE or CD81-PE antibody. Prior to analysis, one μm fluorescent beads (Ultra Rainbow beads, Spherotec GmbH, Lake Forest, IL, USA) were added to each sample at a final concentration of 5 × 10⁴/μL, both as an internal reference for relative size and acquired volume. Each sample was then measured using FL-2 triggering signals for the detection of EVs labeled with PE-conjugated antibodies.

Bone marrow (BM) samples were studied using flow cytometry at the time of diagnosis and relapse and were assessed for MRD presence at follow-up time points with respect to the treatment protocols. The diagnostic panels of fluorochrome-conjugated monoclonal antibodies were based on Moscow–Berlin-group diagnostic standards [40 (link)]. The antibody panels for MRD monitoring were reported previously [41 (link)]. The flow cytometry data were collected on FACSCanto II (Becton Dickinson, San Jose, CA, USA), Navios (Beckman Coulter, Indianapolis, IN, USA) and CytoFLEX (Beckman Coulter) flow cytometers and then analyzed using Kaluza 2.1 software (Beckman Coulter). EuroFlow guidelines for machine performance monitoring were used [42 (link)]. Cytometer Setup and Tracking Beads (BD), Flow-Check Pro Fluorospheres (Beckman Coulter) and CytoFLEX Daily QC Fluorospheres (Beckman Coulter) were used for daily cytometer optimization.

All 50–100 uL of blood was added to the appropriate tubes, and cells were processed according to the manufacturer´s instructions. The antibody panels used were the following:

Lymphocyte Phenotyping panel (DuraCloneTM, Beckman Coulter Life Sciences, Indianapolis, IN, USA): CD16, CD56, CD19, CD14, CD4, CD8, CD3, and CD45 antibodies.

Regulatory T Cells panel (DuraCloneTM, Beckman Coulter Life Sciences): CD45RA, CD25, CD39, CD3, CD45, CD4, Helios and intracellular Foxp3 antibodies.

Myeloid-derived Suppressor Cells (MDSC) panel (DuraCloneTM, Beckman Coulter Life Sciences): CD45, HLA-DR, CD14, CD33, and CD11b antibodies.

All flow cytometry data were acquired on a 10-color/3-laser Gallios flow cytometer (Beckman Coulter). The instrument has not been altered. The stability of the flow cytometer was assured through a quality control procedure using Flow-Check Pro Fluorospheres (Beckman Coulter). According to the manufacturer’s instructions, a compensation matrix for each panel was created using the compensation tubes supplied with each panel. A minimum of 100,000 leucocytes for lymphocyte phenotyping, 100,000 leucocytes for MDSC analyses, and 40,000 leucocytes for regulatory T cells analysis were established [22 (link)]. Data were manually analyzed using FlowJo software v. 10.5 (Tree Star Inc., Ashland, OR, USA).

At passage four, eUCB-MSCs were analyzed by flow cytometry using a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) with a panel of antibodies (Table 1). Flow-check Pro Fluorospheres (Beckman Coulter, Brea, CA, USA) were used to verify instrument optical alignment and fluidics. Flow-Set Pro Fluorospheres (Beckman Coulter, Brea, CA, USA) were used to verify instrument sensibility. eUCB-MSCs were harvested, washed, and resuspended in PBS at a density of 10⁶ cells/mL. Cell suspensions were incubated with monoclonal antibodies for 30 min at 4 °C in the dark, followed by a wash and, for CD73 and CD90, a secondary antibody incubation at 4 °C for 30 min in the dark. Subsequently, the cells were washed and resuspended in 500 µL of PBS. All primary and secondary antibodies are listed in Table 1. The respective mouse isotype antibodies served as controls. A minimum of 20,000 events were acquired for each antibody using Gallios software (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

The optical alignment and fluidics stability of the flow cytometer were tested with Flow Check Pro Fluorospheres (Beckman Coulter, Brea, USA) daily before measurements. Immuno-Trol™ (Beckman Coulter, Brea, USA) was used and evaluated for verification. Flow set Fluorospheres (Beckman Coulter, Brea, USA) were used to set up the photomultiplier tube values weekly. In addition, stained Cyto-Comp cells (Beckman Coulter, O'Callaghan's Mills, Ireland) were used to compensate for the fluorescence overlap.