Cd44 microbeads

All flow-cytometry analyses were performed at the Lerner Research Institute Flow Cytometry core. Cells were processed in flow buffer (0.5% BSA, 2 mM EDTA in PBS) and DAPI-negative viable cells were sorted on a BD FACSAria II. CD44-fluorescein isothiocyanate (FITC) (BD Biosciences, #555478) antibody was used to stain CD44. Enrichment of CD44-positive cells was performed by magnetic labeling of the cells with CD44 microbeads (Miltenyi Biotech, #130-095-194) followed by separation on a MACS column (Miltenyi Biotech).

MDA-MB231 cells were starved for 24 h in DMEM/F12 without FBS and dissociated into single-cell suspensions using 2.5% trypsin followed by centrifugation at 300 g for 5 min. Cell pellets were resuspended in 40 μl of PBE suspension buffer (for approximately 1× 10⁷ cells) and incubated with CD24 microbeads (CD24 Microbead Kit; Miltenyi Biotec, Bergisch Gladbach, Germany) followed by a magnetic separation. CD24⁻ cells were collected, washed, and incubated with CD44 microbeads (Miltenyi Biotec) at 4°C for 15 min. After washing and resuspension in 500 μl of PBE buffer, magnetic separation was used for enrichment of CD44⁺/CD24⁻ cells. To detect the effect of IL-6 on STAT3 signaling in BCSCs and non-BCSCs, before isolation MDA-MB-231 cells were cultured in DMEM for 24 h and then cultured again in DMEM/F12 containing 20 ng/ml of IL-6.

CD44+ cells were isolated from FaDu and Cal27 cells using the CD44 MicroBeads (Miltenyi Biotec, Germany). After magnetic labeling and magnetic separation with LS columns and LD columns, cells were then sorted by using MidiMACS^™ separator (Miltenyi Biotec, Germany), stained with CD44-PE antibody (CST, USA), and analyzed by flow cytometry (BD Biosciences, USA). The stably transfected FaDu and Cal27 cells were collected and resuspended in 100 μl of phosphate-buffered saline (PBS) containing 20 μl FcR blocking reagent (Miltenyi Biotec, Germany), cells were also stained with CD44 antibody for 15 min in the dark, and CD44 expression was detected by flow cytometry.

CD133⁺ and CD44⁺ cells are known to express a high variety of stemness genes. Furthermore, the combined analysis of putative co-CSC markers CD133 and CD44 seems to be more reliable as target biomarkers of low- and high-risk cases of CRC, as compared to single-marker analyses (37 (link)). To delineate the expressions of KDM2B and EZH2 on those co-CSC markers, CD133⁺/ CD44⁺ or CD133⁻/ CD44⁻ cells were isolated from HT-29 cells and separated using a magnetic column included in the MicroBead kit according to the protocol from the manufacturer CD133 MicroBead kit (cat. no. 130-091-895) and CD44 MicroBead kits (cat. no. 130-095-194); both were purchased from Miltenyi Biotec. Averages of 1 × 10⁷ HT-29 cells were centrifuged, and the supernatant was discarded. CD133 microbeads were added and incubated with the cells. Prior to sorting, the column was placed in a magnetic field and rinsed, and the cells were then loaded onto the column (LD Columns, LS Columns, cat. no. 130-042-901, and cat. no. 130-042-401). The acquired CD133⁻ or CD133⁺ cells were incubated with CD44 microbeads (Miltenyi Biotec) and stained with antibodies at 4°C for 15 min. Cells were again washed and magnetically separated.

CD44 positive prostate cancer cells were isolated by MACS using CD44 MicroBeads (Miltenyi Biotech, San Diego, CA; 130–095–194) and probed for TF-Ag expression using anti-TF-Ag antibody JAA-F11 and goat anti-mouse Alexa Fluor 594 conjugated antibody (Molecular Probes by Life Technologies, Thermo Fisher Scientific, Waltham, MA; Cat # A11020). For Western blot analysis, see complete list of antibodies used in Supplementary Methods.

3×10⁴ sorted thymic CD64⁻MHCII⁺CD11c⁺ dendritic cells, thymic, or peritoneal macrophages were cultured in 96-well round-bottom culture plate in 100 μL cDMEM at 37°C in 5% CO₂ incubator for 3 hr to attach. Splenocytes from OT2 mice were stained with biotinylated antibodies to CD8a, CD11b, CD11c, B220, and MHCII (all from BioLegend), washed, and labeled with anti-biotin microbeads (Miltenyi) plus CD44 microbeads (Miltenyi) in cRPMI. The cells were separated on MACS LS columns (Miltenyi) according to the manufacturer’s instructions. Enriched cells (naïve CD4 T cells) were stained with 10 µM CFSE (Sigma) for 5 min in PBS at 37℃ and cocultured with the sorted thymic MHCII⁺CD11c⁺ dendritic cells, thymic, or peritoneal macrophages, in the presence or absence of 0.5-mg/mL OVA protein (Sigma) in cRPMI at 37°C in 5% CO₂ incubator for 72 hr. The cells were collected and stained with antibodies to TCRβ and CD4 (from BioLegend) for flow cytometry analyses of CFSE dilution. The data were analyzed with FlowJo’s Proliferation Modeling module (BD Biosciences).