Anti cd44 pe cy5

Cells were fixed in 70% ethanol and stained in PBS containing 0.1% Triton X-100, 50 μg/mL RNase, and 50 μg/mL propidium iodide. Samples were analyzed by, flow cytometry and the hypodiploid peak constituted the apoptotic population in this analysis. DNA content was measured on a FACSort flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and data were analyzed using ModFit software (Verity Software House, Topsham, ME). At least 1x10⁶ cells were analyzed per sample. For Annexin V Labeling, samples were stained with fluorescein-labeled Annexin V and propidium iodide (Trevigen) according to the manufacturer’s protocol. The two Annexin V positive quadrants of the analysis were taken as the apoptotic fraction. Anoikis Assays were carried out as described previously [39 (link)]. To perform the analysis of cell surface markers (CD44+/CD24-, CD49+/CD24-), MCF10A cells were detached with trypsin, washed in blocking buffer (PBS containing 3% FBS), then stained with anti-CD24-PE (BD Biosciences, San Diego, CA, USA) and anti-CD44-PE-Cy5 (BD Biosciences) or anti-CD49-FITC (BD Biosciences) using 1 ml of antibody per 10⁶ cells, and incubated at room temperature for 1 hr. Following incubation, cells were washed twice with 1 ml PBS. Cells were re-suspended in 1 ml PBS and then analyzed.

Mesenchymal stem cells were detached from culture dish using TrypLE™ express (Thermo Fisher Scientific, Waltham, MA) and collected in Eppendorf tubes. The cells were stained for MSC markers using anti-CD44, PE-Cy5 (BD Biosciences, Franklin Lakes, NJ, #553135), anti-CD73, BV605 (Biolegend, San Diego, CA, #127215), anti-CD105, AF647 (Biolegend, San Diego, CA, #120405), and anti-E-selectin/CD62E, PE (BD Biosciences, Franklin Lakes, NJ, #553751) to assess E-selectin levels. The cells were analyzed on a on a FACSAria II cell sorter using FACSDiva Version 6.1.1 (BD Biosciences) or FlowJo Version 7.6.4 (TreeStar) software.

NOD mice were purchased from Beijing HFK Bioscience CO., LTD (Beijing, China). All mice were housed in a specific pathogen-free environment and all animal experiments were approved by the Institutional Animal Care and Use Committee. Traditional Chinese medicine formula, Ginseng and Astragalus granule (GAG), composed of Ginsenosides of it's stem and leaves, Schisandra, Astragalus, Yam, Radix rehmanniae, Ophiopogon japonicus, Poria, Radix trichosanthis, Rhizoma alismatis and Chinese wolfberry, was purchased from Lunan Houpu Pharmaceutical Co. Ltd (Lunan, China). Cyclosporin A (CsA) was purchased from the Guangdong Provincial Hospital of Traditional Chinese Medicine (Guangzhou, China). Anti-CD4-PE, anti-Foxp3-APC, anti-CD8-PE, anti-CD44-PE-Cy5, anti-CD62L-FITC, anti-CD8-FITC, anti-CD122-PE, and anti-PD-1-APC mAbs were purchased from BD Biosciences (BD Biosciences, San Jose, CA) or eBioscience (eBioscience, San Diego, CA).