Avidin pe

Recombinant high molecular weight (HMW, 1.5–1.8 × 10⁶ Da) HA (CPN spol.s.r.o Czech Republic) was supplied by Farmatrade (Argentina). The Km81 anti-CD44 monoclonal Ab (MAb) was kindly provided by Dr. Pauline Johnson (University of British Columbia, Vancouver, Canada). bHABP, 4MU, X-gal and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (USA). Drugs used were imatinib (Novartis, Switzerland) and vincristine (VCR, Filaxis, Argentina). Antibodies (Ab) against Abl (K-12: sc-131), pAkt1/2/3 (Thr308-R, sc-16646-R), Akt1/2/3 (H136, sc-8312), pERK (Tyr204-R, sc-101761), ERK (C14, sc154), and β-actin (C11, sc-1615), horseradish peroxidase-labeled anti-rabbit (sc-2030) and anti-goat (sc-2033) secondary antibodies, and the biotinylated anti-rat (sc-2041) secondary antibody were purchased from Santa Cruz Biotechnology (USA). The avidin-PE was purchased from eBioscience (USA). The [³H]TdR was purchased from Perkin-Elmer (Boston, USA). RPMI 1640, L-glutamine, streptomycin and penicillin were purchased from Invitrogen (Argentina). The Annexin-V-PE Apoptosis Detection Kit I was purchased from BD Pharmingen™ (BD Bioscience, USA).

FACS analysis was performed using routine protocols with a FACS Calibur flow cytometer (BD Immunocytometry Systems, Franklin Lakes, NJ, USA). Antibodies for staining included APC anti-mouse CD11b (Biolegend, San Diego, CA, USA), Alexa 488 anti-mouse F4/80 (Biolegend), biotin anti-mouse Ly6G (Biolegend) and avidin-PE (eBioscience), PE anti-mouse MR (Biolegend). Dead cells were excluded by propidium iodide staining.
For intracellular staining, cells were stained with cell surface makers first, and then washed with PBS. After centrifuge, cell pellets were suspended completely with fixation buffer on ice for 30 min, and then permeabilized with a Permeabilization Buffer (eBioscience). After centrifuge again, cell pellets were washed and stained with PE anti-iNOS (eBioscience) followed by FACS analysis.
To determine ROS production, cells were harvested and labeled with a ROS probe, according to the recommended protocol (Beyotime), followed by FACS analysis. Briefly, the culture medium was removed and the cells were incubated in dark with the redox-sensitive fluorescent dye (DCFH-DA, 10 µM) diluted by serum-free medium at 37°C/5% CO₂ incubator for 30 min. The cells were then washed with PBS and the fluorescent intensity of DCF was detected by FACS. The ROS level was analyzed using the mean fluorescent intensity (MFI).