Cd24 fitc

Anti-human CD63 PE (R-phycoerythrin) and CD24 FITC (fluorescein isothiocyanate), monoclonal antibodies (Abs), were purchased from eBioscience, Inc. CD81 monoclonal antibody (M38) FITC and Total Exosome Isolation Reagent were obtained from Thermo Fisher Scientific, USA. L-MWCNT-60100 (Shenzhen Nanotech Port Co., Ltd) was used in the synthesis of a MoS₂–MWCNT nanostructure according to previous work.^{26,31 (link)} Fluorescence spectra were recorded by using a Tecan microplate reader infinite M200 at the excitation wavelengths of 488 and 494 nm based on the used fluorophores. Phosphate buffered saline, 1X solution (Fisher Scientific, Inc.) and ultrapure water obtained from a Millipore filtration system were used as the dissolving solutions. All the reagents applied in the breast cancer cell (MCF-7) culture including Dulbecco's modification of Eagle's medium (DMEM), 10% fetal bovine serum (FBS), and antibiotics including penicillin and streptomycin were purchased from Thermo Fisher Scientific, USA and the MCF-7 cell line was purchased from the American Type Culture Collection (ATCC cat. no. HB-72). All NTA analyses were performed by using a ZetaView system, Particle Metrix GmbH.

Cells were detached with a 0.025% Trypsin/2.21 mM EDTA solution in PBS, centrifuged and resuspended in cold PBS, and labeled with CD24-FITC (eBioscience 11-0247), CD44-APC (eBioscience 17-0441), and CD49f-PE (eBioscience 12-0495). After labeling, cells were resuspended in 2% PFA for analysis or cold PBS for sorting. Three-color flow cytometric analysis was performed using a FACScanto II flow cytometer (BD Biosciences, San Jose, CA) equipped with an air-cooled 15mW argon ion laser tuned to 488nm. The emission fluorescence of CD24 FITC was detected and recorded through a 530/30 bandpass filter in the FL1 channel. CD49f PE was detected in the FL2 channel through a 585/42 bandpass filter. CD44 APC was detected in the FL3 channel through a 660/20 bandpass filter. List mode data files consisting of 10,000 events gated on FSC (forward scatter) vs SSC (side scatter) were acquired and analyzed using CellQuest PRO software (BD Biosciences, San Jose, CA). Appropriate electronic compensation was adjusted by acquiring cell populations stained with each dye/fluorophore individually, as well as an unstained control. Cell sorting was performed with the FACSaria from BD Sciences. Gates were established based on unlabeled and single labeled cell samples. Both analysis and sorting were performed through the Cytometry Core Shared Resource at the University of Arizona Cancer Center.

PB mononuclear cells (PBMCs) and SF mononuclear cells (SFMCs) were isolated by density gradient centrifugation. To detect the proportion of CD19⁺CD24^hiCD27⁺ B cells and the expression of RANKL in CD19⁺CD24^hiCD27⁺ B cells of PBMCs or SFMCs, the cells were isolated by density gradient centrifugation and then were stained with mouse monoclonal antibodies as follow: CD19-APC/Cy7 (BioLegend, San Diego, CA, USA), CD24-FITC (eBioscience, San Diego, CA, USA), CD27-APC (eBioscience), RANKL-PE (BioLegend). FMO controls were included. Data was acquired on a FACS Arial II flow cytometer (Becton Dickinson, NJ, USA) and analysed using FlowJo software.
To isolate CD19⁺CD24^hiCD27⁺ B cells, PBMCs were stained with mouse anti-CD19-APC/Cy7, CD24-PE (eBioscience), CD27-APC, then the aimed cell populations were sorted by flow cytometry. Sorted CD19⁺CD24^hiCD27⁺ B cells had a purity of > 95%. These sorted cells were subsequently subjected to reverse transcription-polymerase chain reaction (RT-PCR) or osteoclast differentiation assay.