Swine anti rabbit fitc antibody

The polyclonal rabbit-anti-human CerS3 antibody was from Antikörper-online (Aachen, Germany; 1:50). The secondary antibodies were the swine anti-rabbit-FITC antibody (Dako, Hamburg, Germany; 1:30) and the donkey anti-rabbit-Alexa-Fluor 555 antibody (Thermo Fisher Scientific, Darmstadt, Germany). DAPI was from Sigma-Aldrich GmbH (Taufkirchen, Germany). The following specific inhibitors were used: PPARγ antagonist (GW9962, Enzo, Lörrach, Germany), PI3K inhibitor (LY294002; Sigma-Aldrich GmbH), ERK inhibitor (PD98059, New England Biolabs GmbH, Frankfurt, Germany), p38 MAPK inhibitor (SB203580, Sigma-Aldrich GmbH) and NFκB inhibitor (GIV 3727, Merck Millipore, Darmstadt, Germany). Methanolic sodium hydroxide solution, hexane standards, loganic acid, and loganin were from Carl Roth GmbH (Karlsruhe, Germany). Boron trifluoride–methanol complex was from Merck KGaA (Darmstadt, Germany), and standard methyl heptadecanoate was from Sigma-Aldrich GmbH. Orto-phosphoric acid, 85%, Ph. Eur. p.a. grade was supplied by VWR. Amarogentin and gentiopicroside were purchased from Chromadex Inc. (Santa Ana, CA, USA).

Following enzymatic dissociation, cells were stained within flow cytometry tubes with AlexaFluor^®647 anti-mouse Sca-1, PE anti-human/mouse CD44, AlexaFluor^®488 anti-human CD24 or PE/Cy7 anti-mouse CD24, AlexaFluor^®647 anti-human/mouse CD49f antibodies (all from BioLegend, San Diego, CA, USA) or rabbit anti-OCT4 (Abcam, Cambridge; UK), at a 1:100 dilution. Following 30 min incubation at 4 °C, cells were washed in PBS supplemented with 0.2% BSA and 0.01% sodium azide (Sigma Aldrich). Cells incubated with anti-OCT4 antibody underwent a secondary antibody staining with swine anti-rabbit FITC antibody (Dako, Santa Clara, CA, USA) at 1:50 dilution prior to acquisition. For the evaluation of ALDH activity, Aldefluor^TM kit (STEMCELL Technologies, Vancouver, BC, Canada) was used. Briefly, ALDH substrate was administered to cells treated or not with the ALDH inhibitor DEAB within flow cytometry tubes. Following 45 min incubation at 37 °C, 5% CO₂, cells were rinsed in Aldefluor buffer. Cell viability was assessed by evaluating propidium iodide (Sigma Aldrich) incorporation. All data were acquired through CyAn ADP flow cytometer (DakoCytomation, Beckman Coulter, Brea, CA, USA) and data were analyzed using Summit 4.3.02 Build software (Beckman Coulter).