Carboxyfluorescein diacetate succinimidyl ester cfse

Pairs of sdLN (auricular lymph nodes, draining the pinnae) were removed from mice 4 days after the final infection and were processed to a single‐cell suspension prior to culture at 1 × 10⁶ cells/mL in complete RPMI in the presence, or absence, of 50 μg/mL soluble schistosomula antigen preparation (SSAP) 31 for 72 h at 37°C. In vitro proliferation of sdLN cells was assessed through the decrease of carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies, Paisley, UK) stain 4, 32. Briefly, sdLN cells were labelled with 3 μm CFSE prior to in vitro culture with parasite antigen. After 96 h of culture, sdLN cells were labelled with a fixable live/dead marker (Life Technologies) and then with fluorescently labelled anti‐CD3 and anti‐CD4 mAbs (eBioscience, Hatfield, UK); cell proliferation was measured as a decline in CFSE in the labelled cells determined by flow cytometry. Alternatively, proliferation was measured via incorporation of [³H]thymidine (18·5 kBq per well, PerkinElmer, Coventry, UK) by sdLN cells cultured in vitro with SSAP antigen as described previously 4, 32. Comparable results with regard to CD4⁺ T‐cell proliferation in the sdLN have been obtained previously with CFSE and [³H]thymidine incorporation 4, 12.

Trovafloxacin, 7-aminoactinomycin D, cytochalasin D, sertraline, cycloheximide, serotonin and citalopram were obtained from Sigma-Aldrich (MO). Other reagents were obtained as follows: anti-Fas (05-201, 250 ng ml⁻¹, clone CH11, Millipore, MA), annexin V–fluorescein isothiocyanate (FITC; BD Biosciences, CA), TO-PRO-3 (Life Technologies, NY), carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) and GSK 269962 (Tocris bioscience, UK).

FACS sorting was performed with a MoFlo Astrios cell sorter (Coulter Electronics, Brea, CA) after gating CD45^neg/CD44^pos tumor cells with anti-CD45 (1:10, Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-CD44 (1:1000, Abcam , Cambridge, UK) antibodies. For flow cytometry analysis, cells were stained with Live/Dead and anti-CD44 (Abcam), CD45 (Miltenyi), CD117 (1:100, Miltenyi Biotec) and GLUT1 (1:1000, Abcam) antibodies.
Aldehyde dehydrogenase (ALDH) activity was determined by a fluorogenic dye (ALDEFLUOR^® assay; Stem Cell Technologies, Vancouver, Canada), as described elsewhere [33 (link)]. The specific ALDH inhibitor diethylaminobenzaldehyde (DEAB) was used as a control.
To evaluate the proliferation rate, EOC effusion cells were stained with carboxyfluorescein-diacetate-succinimidylester (CFSE; Life Technologies) as described elsewhere [15 (link)], and seeded at 6 × 10⁴ cells/well in RPMI medium. Flow cytometry analysis was performed 72 h later. For autophagic flux analysis, CYTO-ID^® Autophagy detection kit (Enzo, New York, NY) was used according to the manufacturer's instructions. All the analyses were performed with FACS LSRII (BD); data were analyzed with Flow Jo (TreeStar, Ashland, OR).

Bovine serum albumin (BSA), trypsin from porcine pancreas, α-chymotrypsin from bovine pancreas, and histofine were purchased from Nacalai Tesque (Kyoto, Japan). Carboxyfluorescein diacetate succinimidyl ester (CFSE) was from Invitrogen (Carlsbad, CA). Pentobarbital sodium was from Kyoritsu Seiyaku (Tokyo, Japan). Biotin-conjugated anti-Pseudomonas species polyclonal antibody, anti-MHC-II monoclonal antibody (OX-6) and anti-keratin 10 mouse monoclonal antibody (DE-K10) were from Thermo Fisher Scientific (Waltham, MA). Anti-Ki 67 mouse monoclonal antibody (MIB-5) was from Dako (Glostrup, Denmark). VectaStain ABC Kit and biotin-conjugated anti-mouse IgG antibody were from Vector Laboratories (Burlingame, CA), and 3,3′-diaminobenzidine tetrahydrochloride (DAB Tablet) was purchased from Wako Pure Chemical (Osaka, Japan).

Splenocytes were isolated from C57BL/6 mice (8‐12 weeks old). CD3⁺ T cells were selected using a CD3e MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and then labelled with 5 μmol/L carboxy fluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA) for 7 minutes at 4°C. Labelling was terminated according to the manufacturer's protocol. After washing, cells were activated with 50 ng/mL phorbol myristate acetate (PMA) and 1 μg/mL ionomycin (Sigma‐Aldrich) for 16 hours, and then co‐cultured with or without ASCs for 48 hours. Cell division, as indicated by reduction of fluorescence intensity, was analysed by flow cytometry.