Journal: Frontiers in Immunology

Article Title: The Microenvironment in Barrett’s Esophagus Tissue Is Characterized by High FOXP3 and RALDH2 Levels

doi: 10.3389/fimmu.2018.01375

Figure Lengend Snippet: Antibodies for FACS staining.

Article Snippet: CD4 (T-helper cell)-PerCP , clone L200, 1:20 , BD Pharmingen, San Diego, CA, USA.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: The Microenvironment in Barrett’s Esophagus Tissue Is Characterized by High FOXP3 and RALDH2 Levels

doi: 10.3389/fimmu.2018.01375

Figure Lengend Snippet: Lower expression of CD1a in Barrett’s esophagus (BE) is associated with lower numbers of CD3 + CD8 + -cells in tissue. Panel (A) depicts expression of CD1a by RT-PCR performed on a total of: reflux esophagitis (RE) biopsies from inflamed squamous tissue (INFL RE, n = 9), non-inflamed squamous esophageal epithelium from RE patients (SQ RE, n = 9), squamous esophageal epithelium from controls (SQ C, n = 12), squamous esophageal epithelium from BE patients (SQ BE, n = 10), BE biopsies (BE, n = 11), duodenal biopsies from BE patients (DUO BE, n = 10), and duodenal biopsies from controls (DUO C, n = 5). CD1a expression was corrected for GAPDH, 2 −ΔCT ± SEM. Panel (B) represents the percentage of CD3 + CD8 + -cells from ex vivo cultures of 7 biopsies from inflamed RE tissue (INFL RE), 6 non-inflamed squamous esophageal biopsies from RE patients (SQ RE), 39 controls (SQ C), 17 squamous esophageal biopsies from BE patients (SQ BE), 19 BE biopsies (BE), 15 duodenal biopsies from BE patients (DUO BE), and 11 duodenal biopsies from controls (DUO C). Each bar represents the mean value ± SEM of the percentage of CD3 + CD8 + -cells in the CD3 + population, determined by flow cytometry. Data were analyzed using the Kruskal–Wallis test (CD1a: p < 0.0001, CD3 + CD8 + : p = 0.0002). p -values from comparing individual groups were obtained by using Mann–Whitney U test in case of individual groups, and Wilcoxon signed rank test for different tissue types from the same patient. Adjustment for multiple comparison was conducted by Benjamini–Yekutieli method. (C) Dot plot of a CD3 + , CD4 + , and CD8 + staining on lymphocytes from ex vivo culture of duodenal tissue from BE patient. Cells were gated first on a forward sideward scatter plot and for CD3.

Article Snippet: CD4 (T-helper cell)-PerCP , clone L200, 1:20 , BD Pharmingen, San Diego, CA, USA.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Reflux, Ex Vivo, Flow Cytometry, MANN-WHITNEY, Staining

Journal: Frontiers in Immunology

Article Title: The Microenvironment in Barrett’s Esophagus Tissue Is Characterized by High FOXP3 and RALDH2 Levels

doi: 10.3389/fimmu.2018.01375

Figure Lengend Snippet: Similar CD11c expression in Barrett’s esophagus (BE) and duodenal tissues coincides with higher numbers of CD3 + CD4 + -cells. Panel (A) depicts expression of CD11c by real-time-PCR performed on a total of: biopsies from inflamed tissue in reflux esophagitis (RE) patients (INFL RE, n = 9), non-inflamed squamous esophageal epithelium from RE patients (SQ RE, n = 9), squamous esophageal epithelium from controls (SQ C, n = 12), squamous esophageal epithelium from BE patients (SQ BE, n = 10), BE biopsies (BE, n = 11), duodenal biopsies from BE patients (DUO BE, n = 10), and duodenal biopsies from controls (DUO C, n = 5). CD11c expression was corrected for GAPDH, 2 −ΔCT ± SEM. Panel (B) represent the percentage of CD3 + CD4 + -cells from ex vivo cultures of 7 biopsies from inflamed RE tissue (INFL RE), 6 non-inflamed squamous esophageal biopsies from RE patients (SQ RE), 39 controls (SQ C), 17 squamous esophageal biopsies from BE patients (SQ BE), 19 BE biopsies (BE), 15 duodenal biopsies from BE patients (DUO BE), and 11 duodenal biopsies from controls (DUO C). Each bar represents the mean value ± SEM of the percentage of CD3 + CD4 + -cells in the CD3 + -population, determined by flow cytometry. Data were analyzed using the Kruskal–Wallis test (CD11c: p = 0.05, CD3 + CD4 + : p = 0.0002). p -Values from comparing individual groups were obtained by using Mann–Whitney U test in case of individual groups, and Wilcoxon signed rank test for different tissue types from the same patient. Adjustment for multiple comparison was conducted by Benjamini–Yekutieli method.

Article Snippet: CD4 (T-helper cell)-PerCP , clone L200, 1:20 , BD Pharmingen, San Diego, CA, USA.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reflux, Ex Vivo, Flow Cytometry, MANN-WHITNEY

Journal:

Article Title: Increased inducible apoptosis in CD4 + T lymphocytes during polymicrobial sepsis is mediated by Fas ligand and not endotoxin

doi: 10.1046/j.1365-2567.1999.00765.x

Figure Lengend Snippet: (a)–(h) illustrates the results of typical three-colour flow cytometric analysis of a cell suspension obtained from spleen 24 hr following Sham-CLP (a,c,e,g) or CLP (b,d,f,h) and stimulated in vitro for 24 hr with 2·5 μg Con A/ml. The contour plot (a, b) of the total cell sample illustrates the typical cell cycle progression and the primary gate established on this population (R1). (c) and (d) are the cell cycle histograms of cell number versus DNA content generated from the R1 gated population in (a) and (b) respectively, which show the typical regions defined for these studies. With respect to the Sham, it can be seen that spleen cells extracted from a septic mouse at 24 hr exhibited a population of cells trailing off with lower DNA content, Ao+ (M1), from the G0/G1 peak (M2) as opposed to S/G2/M phase cells (M3)(a, c versus b, d). However, no marked shift in the phenotypic make-up of the stimulated splenocytes is evident in septic animal cells at 24 hr (e versus f). Histograms of DNA content produced from each of these gated phenotypic populations illustrate that the majority of the increase in apoptotic cells is present in the single-positive CD4+ stained cell population (g versus h).

Article Snippet: Cell staining and flow cytometric analysis In an attempt to correlate the changes in the percentage of A o + cells with their phenotypic expression, samples were stained with the combination of antibodies conjugated to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) and the DNA dye 4′,6-diamino-2-phenylindole dihydrochloride (DAPI, Molecular Bioprobes Inc., Eugene, OR) for cell cycle analysis according to the methods of Telford et al. 19 In a typical staining protocol 2×10 6 cells were incubated with 10 μg non-specific mouse immunoglobulin G (IgG)/ml phosphate-buffered saline (PBS) containing 1·0% bovine serum albumin and 0·1% sodium azide (PBS–BSA–Az buffer) for 15 min at 4°, washed by centrifugation, and incubated for 45 min at 4° with 25 μl of PBS–BSA–Az buffer containing 2 μg of FITC-conjugated monoclonal antibody against the murine T helper cell marker, CD4 (clone RM4-5, rat IgG2a) obtained from Pharmingen Inc., San Diego, CA.

Techniques: In Vitro, Generated, Produced, Staining

Journal:

Article Title: Increased inducible apoptosis in CD4 + T lymphocytes during polymicrobial sepsis is mediated by Fas ligand and not endotoxin

doi: 10.1046/j.1365-2567.1999.00765.x

Figure Lengend Snippet: Neither (a) ex vivo (innate or unstimulated) nor (b) in vitro Con A-stimulated (24 hr) splenocytes harvested from mice 24 hr following CLP or Sham-CLP exhibit marked changes in their phenotypic make-up. (c) Neither the mixed (inset) nor those cells differentiated by their phenotypic (CD4 and/or CD8) expression from the ex vivo splenocytes harvested from mice 24 hr following CLP or Sham-CLP exhibited a marked increase in the percentage of Ao+ based on number of cells residing in the M1 region of the cell cycle figures depicted in Fig. 2(c) or (d). Phenotype and associated cell cycle analysis is gated as depicted in Fig. 2(e, g),(f, h). (d) Alternatively (inset) the mixed splenocytes harvested from septic mice show a significant increase (*P < 0·05 versus Sham group) in the percentage of cells which are Ao+ following 24 hr stimulation with Con A. Furthermore, when the CD4 and/or CD8 phenotypic expression was correlated with the extent of Ao+ expression, a marked increase (*P < 0·05 versus Sham group) in the percentage of single-positive CD4+, T helper cells, which were also apoptotic (Ao+) was observed. Mean ± SEM; n is six mice sampled/group.

Article Snippet: Cell staining and flow cytometric analysis In an attempt to correlate the changes in the percentage of A o + cells with their phenotypic expression, samples were stained with the combination of antibodies conjugated to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) and the DNA dye 4′,6-diamino-2-phenylindole dihydrochloride (DAPI, Molecular Bioprobes Inc., Eugene, OR) for cell cycle analysis according to the methods of Telford et al. 19 In a typical staining protocol 2×10 6 cells were incubated with 10 μg non-specific mouse immunoglobulin G (IgG)/ml phosphate-buffered saline (PBS) containing 1·0% bovine serum albumin and 0·1% sodium azide (PBS–BSA–Az buffer) for 15 min at 4°, washed by centrifugation, and incubated for 45 min at 4° with 25 μl of PBS–BSA–Az buffer containing 2 μg of FITC-conjugated monoclonal antibody against the murine T helper cell marker, CD4 (clone RM4-5, rat IgG2a) obtained from Pharmingen Inc., San Diego, CA.

Techniques: Ex Vivo, In Vitro, Expressing, Cell Cycle Assay