Anti biotin pe

Huh7 and Mahlavu cells were seeded onto 100 mm culture dishes. The next day cells were treated with compound 7b, sorafenib at their IC₅₀ concentrations or their combinations, and corresponding DAPT (positive), or DMSO (vehicle) controls. Fluorescence labeling of LCSCs was done using primary antibodies against CD133 (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany cat. no. 130-090-664), and anti-biotin-PE (Miltenyi Biotec, cat. no. 130-090-756), EpCAM (Miltenyi Biotec, cat. no. 130–080-301), or CD90 (Miltenyi Biotec, cat. no. 130-095-403) for flow cytometry analysis as described previously^{36 (link)}. Mouse-IgG-FITC (Miltenyi Biotec, cat. no. 130–092-213), and mouse-IgG-biotin antibodies (Miltenyi Biotec, cat. no. 130-093-018) were used as isotype controls. DAPT (Notch pathway inhibitor) was used as a positive control for cancer stem cell inhibition. Results for each treatment group were compared to that of the DMSO control. Changes in the positivity of CD133 + /EpCAM + cells or CD90 + cells were indicative of enrichment or reduction of the LCSC population. BD Accuri C6 and Novoctye flow cytometer (ACEA Biosciences; Agilent Technologies) were used for flow cytometric analysis.

Large-scale manufacturing of CAR-T cells on CliniMACs Prodigy was carried out under GMP-like conditions into Gene-Cell Therapy clean rooms of the Cell Therapy Unit of Hospital Universitario Reina Sofía (Córdoba, Spain). Two different aphereses from a healthy donor were thawed, and around 100 × 10⁶ (link) T cells were inoculated into the CliniMACs prodigy bioreactor (Miltenyi Biotec). CD4 and CD8 cells were selected with CD8 and CD4 Reagent (Miltenyi Biotec), cultured with IL-7 and IL-15 (Miltenyi Biotec), and activated with αCD3 andαCD28 GMP T cell TransAct (Miltenyi Biotec). On day 2 of the process, these cells were transduced with AWARI-LVs (MOI = 5). Cells were cultured in TexMACs GMP medium containing GMP-grade IL-15 and IL-7 (Miltenyi Biotec) for 9 or 10 days. Final product was collected with 100 mL of NaCl 0.9% + 0.5% human serum albumin (HSA).
Cells were stained with CD3-APC, CD4-FITC, CD8-APC Vio770, CD14-PE Vio770, and CD45-Vioblue (Miltenyi Biotec). To assess the efficiency of transduction, CAR-T cells were stained with CD-19 Biotin and anti-Biotin PE (Miltenyi Biotec). Viability was tested by using 7-AAD (Miltenyi Biotec). Phenotype was determined with CD45RA-APC (HI100) and CCR7-BV421 (2-L1-A RUO) from BD Pharmingen. Cells were acquired on a MACsQuant cytometer (Miltenyi Biotec) and analyzed with MACsQuantify Analysis Software (Miltenyi Biotec).

A four-color flow cytometry panel was designed using a commercial CD19 CAR Detection Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). The reagent consists of a biotinylated CD19 antigen that specifically binds CD19-targeted CARs. In a second incubation step the biotin-labeled CAR T cells are then stained with a fluorochrome-conjugated anti-biotin antibody.
In brief, 200 µl whole blood were treated for 10 min with 2 ml of NH₄Cl-based erythrocyte lysing solution (Beckman Coulter, Krefeld, Germany) and washed with PBS, containing 0.5% HSA. After removal of the supernatant down to 200 µl, cells were resuspended and 100 µl were transferred to a new flow cytometry tube. Following 15 min of incubation with 1 µl CD19 CAR Detection Reagent, cells were washed twice and incubated for 15 min with 1 µl Anti-Biotin-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), 10 µl 7-AAD, 5 µl CD3-APC, and 5 µl CD45-KrO (all purchased from Beckman Coulter Immunotech, Marseille, France). After a final washing step, cells were acquired on a NAVIOS flow cytometer (Beckman Coulter, Krefeld, Germany). Cellular debris was excluded based on light scatter properties and CAR T cells were defined as 7-AAD^-/CD45⁺/mononuclear cells/CD3⁺/CD19 CAR⁺ (Supplementary Figure 1).

Whole blood was removed by retroorbital bleed and stained for flow cytometry (BD FACSCanto II) using the following antibodies or fluorescent dyes: Biotin anti-mouse CD71 REAfinity (130-109-572, Miltenyi), anti-biotin-PE (130-113-291, Miltenyi), 0.0025 µg/µL PE-Cyanine7 Anti-Mouse TER-119/Erythroid Cells (116222, BioLegend), 0.0025 µg/µL APC-Cyanine7 Anti-Mouse CD45 (103116, BioLegend), 100 nM Thiazol orange (390062, Sigma Aldrich), 500 nM MitoTracker Deep Red FM (8778, Cell Signaling), and 2.5 µg/mL 7-AAD live-dead viability staining (420404, BioLegend).