Cfda se

To evaluate whether membrane damage was linked to cell leakage of intracellular components, microbial cells from an overnight culture were washed and diluted in sterile filtered (0.22 μm) phosphate-buffered saline (PBS) (NaCl 8 g/L; KCl 0.2 g/L; Na₂HPO₄ 1.44 g/L; KH₂PO₄ 0.24 g/L; pH 7.4) to a final concentration of 3 × 10⁹ cells per mL. The cell suspension was supplemented with 4 μM cFDASE (Sigma-Aldrich), which is a precursor molecule of cFSE^{32 (link)}. Then, the cell suspension was exposed to each antimicrobial (100 μg/mL) or to chlorhexidine (100 μg/mL) (Sigma-Aldrich) at 30–37 °C (according to the optimum temperature of growth). As a control, the cFSE-labeled cell suspension was also exposed to a volume of DMSO equal to that used for all tested molecules. After 30 min of incubation, 1 mL of sample was used to measure cFSE fluorescence cell leakage. Briefly, the sample was centrifuged (13000 rpm, 2 min), and the cell-free supernatant was transferred to a 96-microtiter plate for the measurement of cFSE fluorescence in a Victor 3 fluorometer (PerkinElmer)^{32 (link),33 (link)}. The fluorescence data were calculated as the average of three independent assays and expressed as arbitrary units of fluorescence ± standard deviation.

Cell cycle analysis of LN T cells was performed using Coulter Reagents Kit (Beckman-Coulter, Villepinte,France) on the basis of DNA staining with propidium iodide (PI) as described [27 (link)]. At least 30000 events were analyzed at low speed (100 events/second) and collected on list mode files. The percentage of T cells in the different phases of the cell cycle (i.e. G0/G1, S, G2/M phase) was determined. T lymphocyte proliferation capacity was analyzed after 24, 48 and 72H of culture of T cells with plate bound anti-CD3- (0.1/-1-2 μg/ml, as indicated in figure legend) with or without recombinant anti-CD28 (2μg/ml), with or without recombinant IL-2 (50U/mL) by enumeration in a haemocytometer after dilution of T cells in trypan blue, or by cytometric analysis of stimulated LN T cells labeled with the fluorescent dye carboxyfluorescein diacetate succinimidylester (CFSE) (CFDASE, Sigma-Aldrich, St Quentin Fallavier, France). Propidium iodide staining [1μg/ml] was performed in CFSE labelled cells [27 (link)]. In each histogram, the percentage of the dividing cells per cell generation was determined by quantification of CSFE cellular fluorescence halving using a flow cytometer CyAn (Beckman-Coulter, Villepinte, France), and data were analyzed using FlowJo software (v10.0.6 Tree Star, Ashland, OR).

To determine the effect of bezafibrate on T cell proliferation in the RA model, spleen, axillary, and popliteal lymph node cells from naïve, bezafibrate-treated, and non-treated controls subjected to AIA were stained with 5 μM CFDA-SE (Sigma-Aldrich) by incubation for 5 min in the dark at RT. Stained cells were washed five times with FACS buffer (PBS + 1% FBS). Stained cells (2 × 10⁶/mL in a total volume of 200 μL) were stimulated with mBSA (50 μg/mL) and cultured at 37 °C in 5% CO₂ and 95% humidity. After 72 h, cells were harvested and analyzed for diminished CFSE-stain by FACS; see gating strategy (Additional file 1: Figure S11) CFSE-stained cells from a naïve mouse were used to define non-proliferating cells [50 (link)]. Assessment of antigen recall responses of CD4+ T helper cells among spleen and lymph node cells showed that the systemic intraperitoneal treatment with bezafibrate that protected from arthritis also inhibited proliferation of CD4+ T helper cells, which was not the case for bezafibrate treatment locally or at sensitization (Additional file 1: Figure S10). Thus, the protective effect of bezafibrate on arthritis development is contingent on its ability to inhibit T helper cell proliferation.

Further, 1 × 10⁵ Treg cells were suspended in 10 µL of PBS and were injected subconjunctivally to alkali-injured mice using a 33-gauge metal needle and a 50-µL syringe (Hamilton Company, Reno, NV, USA). Mice receiving 10 µL of PBS served as the control. In in vivo tracing of the dynamic migration of Tregs, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (Sigma Aldrich, St. Louis, MO, USA) was used to stain the sorted Tregs before the injection. According to the protocol of CFDA-SE, the cells were incubated with the dye in the dark at 37°C for 12 minutes, followed by the addition of an equal volume of the culture medium containing 10% fetal bovine serum (Invitrogen) for 5 minutes to quench the excessive dye.

The recombinant lactobacillus pPG-E2-DCpep/LC W56 was cultured overnight in MRS broth at 37 °C and were centrifuged at 12,000× g for 5 min. After washing twice with sterile PBS, the cells were adjusted to a concentration of 10¹⁰ CFU/mL followed by labeling with 5’-(and 6’)-carboxyfluorescein diacetate succinimidyl ester, cFDA-SE (Sigma) at 37 °C for 25 min. Before oral administration, the labeling efficiency of recombinant lactobacillus with cFDA-SE was analyzed by flow cytometry. After that, one group of 40 mice was orally dosed with 200 μL of approximately 10¹⁰ CFU/mL of cFDA-SE-labeled pPG-E2-DCpep/LC W56 for each mouse, and another group of 40 mice was fed with sterile PBS as control. On days 1, 3, 5, 7, 9, 11, 13, and 15 after oral administration, five mice from each group were selected randomly, and the duodenum, jejunum, ileum, and colon were extracted from each mouse and cut longitudinally followed by removal of any residual food particles or fecal material. Subsequently, after adding 200 μL of PBS to every 1.0 cm of tissue and dislodging microbes from intestinal mucosal surface, the presence of adhering cFDA-SE-labeled pPG-E2-DCpep/LC W56 in each intestinal tract was determined by flow cytometry.

The glyco-code of melanoma tumor cells was assessed by performing the GLYcoPROFILE™ with the LEctPROFILE^® plates from GLYcoDiag (Orléans, France). The assessment of interactions of lectins with glycans on cell surfaces were achieved according to GLYcoDiag’s protocol (36 (link), 37 (link)). When cells grew up to 80–90% confluence in 75 cm² culture flask, cells were washed with PBS and harvested with a Trypsin/EDTA solution. After washing and centrifugation, the cells were suspended in PBS and labeled with carboxyfluorescein diacetate succinimidylester (CFDA-SE, Sigma-Aldrich, St. Louis, MO, USA) in PBS. Next, 100 μL of labeled cells (about 2 × 10⁵ cells) were added in each well of the LEctPROFILE^® plates and incubated 2 h at room temperature under gentle agitation. After washing with PBS, fluorescence intensity was measured using a microplate reader (λ_ex = 485 nm, λ_em = 530 nm, Fluostar OPTIMA, BMG LABTECH, France). In parallel, a calibration curve was achieved with the labeled cells solution to determine the number of cells stayed in interactions with lectins.