Fcs express 6

To determine the effect of the investigated compounds on the heterogeneity of breast cancer cells, flow cytometric studies were carried out with the aim to reveal the changes in the proportion of CSC-like, CD44+/CD24− cells within a heterogeneous population of MCF-7 cells (unsorted, uninfluenced by the CSC-enrichment after FACS sorting). The adherent MCF-7 cells were transferred to ultra-low attachment plastics (ULA; Corning, NY, USA), cultivated in DMEM medium supplemented with B27 (2%), EGF (20 ngmL⁻¹) and HSA (0.15% (w/v)). Cells were cultured for 96 h to form the spheroids and then treated for another 24 h with the investigated compounds at the concentrations corresponding to their respective values of IC_50,72h. The cells were subsequently washed with DPBS and incubated with CSDR to prepare single cells from the preformed spheroids and processed for the staining with anti-CD44-APC and anti-CD24-PE antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer instructions. The heterogeneity of surface markers on MCF-7 cells was determined by using flow cytometry (BD FACSVerse, BD Biosciences, San Jose, CA, USA). The data were analyzed using FCS Express 6 software (DeNovo software; Glendale, CA, USA).

Cells were collected, stained, and analyzed by flow cytometry (BD Cytoflex S flow cytometer) as follows. Staining for membrane surface molecules included use of the following antibodies: EpCAM-PE (phycoerythrin): [VU-1D9] (Abcam) and isotype control [MOPC-21] (BD Pharmingen); E-cadherin-PE: CD324 [DECMA-1] BioLegend) and isotype control [A95-1] (BD Pharmingen); and N-cadherin-APC (Allophycocyanin): CD325 [8C11] (BioLegend) and isotype control [27 (link)–35 (link)] (BD Pharmingen). Staining was performed following standard methods. Cells were stained for 30 min (EpCAM) and 20 min (E-cadherin and N-cadherin). For intracellular staining of CCT2 we used the antibody CCT2-PE: [NP_006422] (LSBio) and the isotype control [MOPC-21] (LSBio), following the method in ThermoFisher Scientific’s “Protocol A: two-step protocol: intracellular (cytoplasmic) proteins” and incubating the antibody for 70 min. When optimizing the CCT2 intracellular stain for CSS Autoprep conditions, we adjusted the antibody staining protocol for CCT2-PE to match the 20 min in the CSS Autoprep automated conditions. All data was generated using FCS Express 6 software (De Novo).

Brain tissue disassociation, nuclei purification with sucrose gradient, and antibody staining were carried out according to previously published protocol.^{54 (link)} Primary antibodies: Olig2- AlexaFluor 488 (1:50 v/v, EMD Millipore MABN50A4, MO) and NeuN-AlexaFluor 647 (1:1000 v/v, Abcam ab190565, MA) were used. Flow cytometry was performed on a BD Melody (BD Biosciences, CA). The data were analyzed by the FCS Express 6 software (De Novo Software, CA). Doublets discrimination analysis was done based on signal processing (height vs. width). Olig2⁺ and NeuN⁺ cells were determined based on the AF488 and AF674 fluorescence, correspondently.

Cells were plated in 6-cm dishes in DMEM/F12 medium that was supplemented with 5% FBS 24 h prior to drug treatment. The cells were then treated with DMSO (control), lapatinib (3 µM), MK2206 (6 µM for PC3-lineage cells, 15 µM for DU145-lineage cells) or both for 72 h. The attached cells were collected by trypsinization, re-suspended in complete medium at a concentration of 1 × 10⁶/mL. Acridine orange (AO) and PI reagents (Nexcelom Bioscience, Lawrence, MA, USA) were added to the cells per the manufacturer’s instructions and loaded into a counting chamber. Both live cells (AO positive, green fluorescence) and dead cells (PI positive, red fluorescence) within the counting chamber were assessed using a Nexcelom Cellometer Fluorescent Viability Cell Counter (Nexcelom Bioscience). The data were analyzed using FCS Express 6 software (De Novo Software, Glendale, CA, USA). SEM was calculated from two independent experiments.