Trypsin edta solution

After harvesting the hES cells using a 0.05% trypsin-EDTA solution (PAA Laboratories, Linz, Austria) and washing them with PBS, single hES cell suspensions were fixed using a 1.6% paraformaldehyde (PFA, Sigma-Aldrich) solution for 10 minutes at room temperature (RT) as we described previously [21 (link)]. The cells were washed and stained using a permeabilisation buffer (Foxp3 Staining Buffer Set, e-Biosciences), blocked using a 2% goat serum (PAA Laboratories) in a permeabilisation buffer (15 minutes) and stained with the appropriate antibodies or their isotype control antibodies for 30 minutes at RT. For cell cycle analysis, the cells were stained with DAPI (Sigma-Aldrich Chemicals). Flow cytometry data were acquired with FACSAria using FACSDiva software (BD Biosciences). Cell permeabilisation, fixation and staining, and data acquisition were done on the same day. The populations that were positive or negative for specific markers were selected using density plots according to the population's borders or using specific biological samples (pluripotent or differentiated hES cells) or specific isotype controls.

After harvesting hES cells with 0.05% trypsin-EDTA solution (PAA Laboratories, Linz, Austria) and washing with PBS, single hES cell suspensions were fixed by using 1.6% paraformaldehyde (PFA, Sigma-Aldrich) for 10 min at RT as described for detection of intracellular phosphoproteins [15 , 16 ]. Cells were then washed and stained using a permeabilisation buffer (Foxp3 Staining Buffer Set, e-Biosciences). Cells were blocked using 2% goat serum (LabAs Ltd., Tartu, Estonia) in a permeabilisation buffer (15 min at RT) and stained with appropriate antibodies or their isotype control antibodies for 30 min at RT. For cell cycle analysis, cells were stained with DAPI (Cystain DNA, Partec GmbH, Münster, Germany). Flow cytometry data were acquired with FACSAria using FACSDiva software (BD Biosciences). In some experiments, after fixation with 1.6% PFA, cells were permeabilized with ice-cold methanol for 20 min at 4°C, washed with PBS containing 1% BSA and 2 mM EDTA, and then blocked and stained with antibodies as described above. Cell permeabilisation, fixation and staining, and data acquisition for all samples were done on the same day. The populations positive or negative for specific markers were selected on density plots according to a population's borders or by using specific isotype controls.

Cells treated with RA or DMSO were harvested with 0.05% trypsin-EDTA solution (PAA Laboratories) and washed with PBS. The single cell suspensions were fixed using 1.6% paraformaldehyde (PFA, Sigma-Aldrich) for 10 min at room temperature (RT). Cells were washed with Permeabilization buffer (Permeabilization buffer, e-Biosciences), blocked using 2% goat serum (PAA Laboratories) in Permeabilization buffer (10 min at RT) and stained with appropriate antibodies or their isotype controls (Table A in S1 File) for 30 min at RT. For cell cycle analysis cells were further stained with DAPI (Sigma-Aldrich). Compensation controls were prepared by using CompBead Plus compensation particles (BD Biosciences) incubated with appropriate antibodies. Flow cytometry data were acquired with FACSAria using FACSDiva software (BD Biosciences) and analyzed with FACSDiva (BD Biosciences) or FlowJo software (FlowJo, LLC). At least three independent flow cytometry experiments were conducted for each antibody combination except for detection of α1, α2 and α3 laminin chains where two independent experiments were performed.

MCF-7 cells were detached, washed and plated in a 6 well low attachment plates (Corning), filled with 3mL MammoCult Basal Medium and supplemented with MammoCult proliferation Supplements, 4 μg/mL Heparin, 0.48 μg/mL Hydrocortisol and penicillin (100 U/mL), streptomycin (100mg/mL). MCF-7 mammospheres were started forming after day 4-6 and were processed at day 10. Secondary generation mammospheres were generated by incubating the primary mammospheres with 1 X Trypsin EDTA solution (PAA Laboratories) for 2-3 min and resuspended in the above medium and plated again in the low attachment plates. After 7 days the capacity of cells to generate secondary mammospheres were tested by visualizing the cells with the inverted light microscope (Axiovert).