Anti pe micro bead

To separate the CD3⁻ cells from other IEL, we employed MACS based cell separation technology. Accordingly, a total of 10⁸ isolated IEL cells/ml were washed with cold PBS/BSA/EDTA at 4°C for 10 min at 400 × g. The pellet was then re-suspended in 100 μl cold PBS/BSA/EDTA and 10 μl CD3 PE (Southern Biotech, USA) and mixed by vortexing. The cells were later incubated on ice and shaken at 100 rpm for 30 min. Then, 1 ml of cold PBS/BSA/EDTA was added and centrifuged at 4°C for 10 min. After that, 100 μl cold PBS/BSA/EDTA and 10 μl Anti PE Micro Bead (Miltenyi Biotec, Germany) were added, vortexed, and incubated on ice for 30 min. The cells were washed again and re-suspended in 1 ml cold PBS/BSA/EDTA. Finally, the MACS column was prepared and, the columns were rinsed with cold PBS/BSA/EDTA and the cells were applied to the columns in a magnetic field. All CD3⁻ cells passed from the columns were collected and put on ice for further characterization.

Isolation of the CSC-like subpopulation was conducted via magnetic-activated cell sorting (MACS) separation, which involved a MACS Column and MACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany). The parental MDA-MB-231 cells (pMDA) were filtered using a 70-µm cell strainer (BD, Franklin Lakes, NJ, USA) to obtain single cell suspension prior to cell sorting. MACS separation was conducted according to the protocol provided by manufacturer. Briefly, the 24- subpopulation was first isolated via CD24 MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) by positive depletion of CD24-expressing cells, followed by positive selection of CD44-expressing cells using anti-CD44-PE (Biolegend, San Diego, CA, USA) and anti-PE MicroBead (Miltenyi Biotec, Bergisch Gladbach, Germany). This resulted in the acquisition of the sorted CD24⁻/CD44⁺ CSC-like subpopulation (sMDA).