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