All samples were acquired by a FACSCanto flow cytometer (Becton Dickinson) and for each sample 20,000 events were acquired and analyzed by FCS 6 Express software (De Novo Software).

To evaluate the DNA index, unfixed DHSF-BR16 cells and human lymphocytes obtained from a health volunteer were stained with a Propidium Iodide (PI) staining technique15. The DNA index was calculated as the ratio of DHSF-BR16 cells and peripheral blood diploid (2n) lymphocytes G0/G1 fluorescence channels. According to Ross et al.16, a DNA index of 1.0 is indicative of diploidy whereas a ratio ranging from 0.85 to 1.9 is indicative of ipo- or hyper-diploidy, respectively.

These growth parameters have been evaluated in the same experiment by cell counting and PI staining, respectively, according to the procedures described in Coronnello et al.17.

The expression of CD44 and CD24 surface antigens was measured after DHSF-BR16 and MCF-7 cell staining with CD44 and CD24 antibodies (Thermo Fisher), according to the manufacturer’s instructions (Supplementary Methods).

ROS production was evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Kodak)18. Fresh stock solution of 2 mM (w/v) DCFH-DA in ethanol was prepared and kept at − 20 °C in the dark until the use. Briefly, cells were incubated with DCFH-DA solution (10 µM) at 37 °C for 1 h in a humidified atmosphere with 5% CO2. After washing, the fluorescence of cells was acquired by FACS (i.e. λex 488 nm, λem 530 nm).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.