Cd44 pe

The antibodies used for Western blotting (WB), immunofluorescence (IF), immunohistochemistry (IHC) and flow cytometry (FC) were as follows: human and mouse Annexin A1 (WB, IF), mouse mAb clone EH17a (Santa Cruz Biotechnology, Dallas, TX, USA). Human Annexin A1 (IHC), mouse mAb clone MRQ-3 (Cell Marque, Sigma Aldrich). Mouse E-cadherin (IF, IHC), mouse mAb clone E-cad/36 (BD Transduction Laboratories, BD Biosciences, Scoresby, Vic., Australia). Mouse CD24 (FACS), CD24-PE-Vio770 or CD24-APC, rat mAb clone M1/69 (Miltenyi Biotec, Macquarie Park, NSW, Australia), mouse CD44 (FACS), CD44-PE, rat mAb clone IM7.8.1 (Miltenyi Biotec), mouse Epcam/CD326 (FACS), Epcam-APC, rat mAb clone G8.8 (eBioscience, Invitrogen, Thermo Fisher Scientific, Scoresby, Vic., Australia). Mouse cytokeratin 14 (IHC), mouse mAb clone LL002 (Abcam, Melbourne, Vic, Australia). Mouse pan-cytokeratin (recognizing type II cytokeratins 1, 5, 6, and 8) (IHC), mouse mAb clone PCK-26 (Sigma-Aldrich). Mouse cytokeratins 8–18 (IHC), rabbit mAb clones EP17/EP30 (Dako, Leica Microsystems, Macquarie Park, NSW, Australia). Mouse alpha smooth muscle actin (αSma) (IF), rabbit pAb ab5694 (Abcam). Mouse Sca1 (Ly6a) (FACS), Sca1-PE, rat mAb clone D7 (Miltenyi Biotec). Mouse vimentin (IHC), rabbit pAb R28 (Cell Signaling Technology, Danvers, MA, USA).

The mesenchymal character of isolated gingival fibroblasts was confirmed by investigating the presence of the following surface markers: CD44, CD90, and CD105 with a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). The following antibodies were used: CD44-PE (human, 130-113-897); CD90-FITC (human, clone: REA897, 130-114-901); CD105-APC (human, clone: REA794, 130-112-324) and REA Control (S)-PE (130-113-438), REA Control (S)-FITC (130-113-437), REA Control (S)-FITC (130-113-437), REA Control (S)-APC (130-113-434) and REA Control (S)-PE (130-113-438) from Miltenyi Biotec (Bergisch Gladbach, Germany). The data were analyzed using CellQuest Pro Software (Becton Dickinson, San Jose, CA, USA, version 5.2.1).

ALDEFLUOR assays (Stem Cell Technologies) to detect ALDH1 activity were performed according to manufacturer’s instructions. Diethylaminobenzaldehyde (DEAB) was used as an ALDH1 inhibitor to set ALDH1 gates. Cell surface levels were determined with anti-human antibodies CD44-PE, CD24-APC, CD133-APC, CD326-FITC, CK18-FITC and CK20-FITC (Miltenyi Biotec). All samples were analyzed on a FACS CANTO II (BD Biosciences) using the FACS DIVA software.

The freshly sorted cells were seeded in non-adherent 24-well plates (1 × 10⁵) and incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU; MilliporeSigma, Copenhagen, Denmark) for 24 h. Subsequently, the cells were fixed with 3.7% formaldehyde for 15 min and permeabilized in 0.5% Triton X-100 in PBS for 20 min. The cells were stained using the Click-iT EdU 488 Proliferation Kit (MilliporeSigma, Copenhagen, Denmark) following the manufacturer’s protocol. Next, cells were washed twice in 3% BSA/PBS and blocked for 1 h in 2% BSA/2% FBS in PBS. Cells were incubated in the dark for 20 min with 150 µL CD44-PE (1:5000) (Miltenyi Biotec) and counterstained with 10 µg/mL DAPI (MilliporeSigma, Copenhagen, Denmark) for 10 min. Cells (200–600) were analyzed using a Leica DM 2000 LED microscope (Leica Microsystems, Copenhagen, Denmark). Quantification was performed manually using the ‘cell counter plugin’ in ImageJ version 1.50i (National Institute of Health, Bethesda, MD, USA).

Media, sera and antibiotics for cell culture were provided by Innoprot (Derio, Bizkaia, Spain) and Gibco (Waltham, MA, USA). MGO (40% in water) was from Sigma-Aldrich (St. Louis, MO, USA). Protein electrophoresis and western blot reagents were from Bio-Rad (Richmond, VA, USA) and ECL reagents from Pierce (Rockford, IL, USA). The antibodies used for western blot are anti-MGO (Abcam, Trumpington, Cambridge, UK) and anti-14.3.3 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-vinculin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling, Technology, Danvers, MA, USA). CD29-APC, CD44-PE, CD45-FITC, CD31-APC, CD90.2-PE and Sca-1-APC antibodies were provided by Miltenyi Biotec (Auburn, CA, USA). TRIzol and SuperScript III were from Invitrogen (Carlsbad, CA, USA). SYBR Green Supermix was from Bio-Rad (Hercules, CA, USA). All other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).

50 000 viable B16-F10 cells (trypan blue exclusion test) dissociated from one-week-old tumorspheres or from trypsinized adherent monolayers were incubated for 30 minutes at 4°C in the dark with 0.5 µg of rat anti-mouse monoclonal CD133-APC, CD44-FITC or CD24-PE antibodies (eBiosciences, Paris, France) in 100 µL of a buffer solution consisting in PBS containing 3% bovine serum albumin (Sigma). Immunostaining was also performed for HT-29, MCF-7 and MDA-MB-231 cell lines using mouse anti-human monoclonal CD133-APC, CD44-PE, CD44-APC or CD24-FITC antibodies (Miltenyi Biotec, Paris, France) according to the manufacturer’s instructions. Cells were then washed and analyzed with an Accuri C6 flow cytometer (BD biosciences, USA).