Accuri c5

Each cell was collected and washed with flow cytometry buffer (PBS with 2% FBS and 0.1% NaN3). 2 × 10⁵ cells were stained with the primary antibody: an anti-canine CD20 antibody (4E1-7, 4E1-7-B, 4E1-7-C, or 1E-4-B), a PE-labeled anti-CD21 antibody (Bio-Rad Laboratories, Inc., Berkeley, CA), or an isotype control (rat IgG_2a, BioLegend, San Diego, CA, USA), followed by a secondary antibody: PE-labeled anti-rat IgG (SouthernBiotech, Birmingham, AL), Dylight 649-labeled anti-rat IgG (BioLegend), or Alexa 647-labeled anti-dog IgG (Jackson ImmunoResearch, West Grove, PA, USA).
To determine the subclass of 4E1-7, NRK/cCD20 cells were first stained with the anti-canine CD20 antibody (4E1-7), and then stained with either biotin-labeled anti-rat IgG-κ, anti-rat IgG-λ, anti-rat IgG1, anti-rat IgG_2a, or anti-rat IgG_2b antibody, followed by incubation with streptavidin-PE (Thermo Fisher Scientific Inc.). Results obtained using a BD Accuri C5 flow cytometer (BD Biosciences) were analyzed using FlowJo v.10 software (Tree Star Inc., Ashland, OR, USA).
Binding affinity of each antibody was determined by flow cytometry. Each cell was stained by the serially diluted antibody, as described above. Kd value was determined using the Nonlinear Regression Michaelis–Menten curve fit by JMP14.0 software (JMP Japan, Tokyo, Japan).

To determine the bacterial membrane permeability, herein, we used propidium iodide (PI) staining assay following the reported procedure [40 (link),41 (link)]. Briefly, the bacterial broth was initially diluted to 8 × 10⁷ CFU/mL and treated with the designed nanocomposites (20 µg/mL), in addition to the negative as well as positive control groups, of media treatment and 70% isopropanol, respectively. After 6 h of incubation, the PI stain (20 µg/mL) was added to the bacterial suspension and incubated for a further 15 min. The bacteria culture was washed twice and suspended in PBS. Finally, the fluorescence of PI was measured (λ_Ex/Em -536/623 nm) within 2 h using the flow cytometer (BD Accuri-C5).

For the drug efflux assay, C2C12 myotubes were incubated with CT-26 cell conditioned media and SFI for 6, 12, and 24 hours. For the apoptosis analysis, the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA, United States) was implemented to stain the cells. To measure the reactive oxygen species, the cells were stained with a fluorescein-based general ROS indicator 2′, 7′-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, bestBio, China) for 30 min at 37°C and 5% CO2. For mitochondrial membrane potential analysis, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine (JC-1, bestBio, China) was utilized to measure the cells. All flow cytometry analyses were performed with BD Accuri C5 or LSRFortessa and analyzed with the FlowJo software.

Chemical hypoxia was induced with 100 μmol/mL CoCl₂ on the two types of spheres for 2 h prior to X-ray. Under hypoxic conditions, cell cycle distribution, apoptosis rates, and ROS levels in each group were detected by FCM using BD Accuri™ C5 (BD Biosciences).

MCF-7 and MDA-MB-231 cells were obtained from ATCC (Manassas, VA, USA). They were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 1% P/S (Gibco, USA) in an incubator with 5% CO₂ at 37°C. Then, according to the culture method reported by Dontu et al. [19 (link), 20 (link)], the two kinds of breast cancer cells were plated in ultralow attachment 6-well plates with SFM including DMEM-F12 (Gibco), P/S, bFGF, EGF, B27 (Cyagen, USA), insulin, and BSA (Sigma-Aldrich). The growth medium was replenished every 2 days with fresh medium. Cells cultured under the condition forming nonattached tumor spheres were digested once a week. Third-generation spheres were collected to detect the CD44⁺CD24^-/low cells by FCM (BD Accuri™ C5, USA).