Journal: bioRxiv

Article Title: Haem toxicity in the aged spleen impairs T-cell immunity through iron deprivation

doi: 10.1101/2024.05.05.592551

Figure Lengend Snippet: (A-D) (A) Scheme of experimental design. Naïve T cells (CD4+CD25-CD62L+CD44lo) were purified from the spleen or peripheral lymph nodes (LNs) of aged mice (20-22 months old). The cells were kept in two separate pools and stimulated ex vivo for 48 hr using plate bound anti-CD3/anti-CD28. (B) Cell viability, analyzed by flow cytometry. (C) Quantitation (D) a representative FACS histogram demonstrating cell proliferation, detected by dilution of CellTrace Violet. (E) Quantification, and (F) a representative FACS histogram showing CD39 expression on T cells isolated from the spleen and LNs of young (8 weeks old) and aged mice, analyzed by flow cytometry. (G) Scheme of experimental design for T cell adoptive transfer. Young T cells derived from TdTomato+ transgenic mice were transfused into young or aged C57Bl/6 wild type recipients. After 2-3 weeks, recipient mice were sacrificed, and CD4+ T cells purified from the spleen and LNs for analysis of (H) cell size and (I) CD39 expression by flow cytometry. (J, K) A portion of the cells were loaded with CellTrace violet, and stimulation for 48 hr, to assess proliferation. Each data point represents an individual mouse. (*p<0.05, **p<0.01; paired student’s t test when comparing cells derived from spleen and LNs of the same mouse, and unpaired student’s t test when comparing values across age groups). Each panel shows representative data of at least 2 independent experiments.

Article Snippet: For isolating naïve T cells from aged mice, biotinylated anti-mouse/human CD44 (103004, biolegend), and biotinylated anti-mouse CD25 (130-049-701, Miltenyi Biotech) were added to the company’s premade antibody cocktail for negative selection of naive T cells, followed by a positive selection of CD62L + naïve T cells on a magnetic column.

Techniques: Purification, Ex Vivo, Flow Cytometry, Quantitation Assay, Expressing, Isolation, Adoptive Transfer Assay, Derivative Assay, Transgenic Assay

Journal: Journal of Ginseng Research

Article Title: Effect of polysaccharides from a Korean ginseng berry on the immunosenescence of aged mice

doi: 10.1016/j.jgr.2017.04.014

Figure Lengend Snippet: Effect of APGP on age-correlated CD11c-positive B cells and on T cell subpopulations in aged mice. (A) Total splenic cells (5 × 10 6 cells/mL) isolated from old and young mice administered the indicated doses of compounds were analyzed by flow cytometry using antibodies specific for CD11c and B220, a marker of B cells. (B) The population of age-correlated CD11c-positive B cells in total splenic cells was measured and plotted. (C) Total thymic cells (5 × 10 6 cells/mL) isolated from old and young mice administered the indicated compounds were analyzed by flow cytometry using antibodies specific for CD4, a marker of helper T cells, and CD8, a marker of cytotoxic T cells. The populations of (C, left panel) CD4-positive helper T cells and (C, right panel) CD8-positive cytotoxic T cells in total thymic cells were measured and plotted. (D) Total splenic cells (5 × 10 6 cells/mL) isolated from old and young mice administered the indicated compounds were analyzed by flow cytometry using antibodies specific for Foxp3 and CD25, markers of activated Treg cells. (E) The populations of Foxp3-positive Treg cells and activated double-positive (Foxp3 and CD25) Treg cells in total splenic cells were measured and plotted. Data are presented as the means ± SD of one biological experiment performed with six technical replicates ( n = 6). * p < 0.05, ** p < 0.01 compared to a control group. APGP, acidic-polysaccharide-linked glycopeptide; NK, natural killer; PSK, polysaccharide-K; SD, standard deviation; Treg, regulatory T cells.

Article Snippet: Fluorochrome-labeled monoclonal primary antibodies specific for CD-11c (CD11c-PE), NK1.1 (NK1.1-APC), B220 (B220-APC), Foxp3 (Foxp3-PE), and CD25 (CD25-biotin) and streptavidin–fluorescein isothiocyanate for fluorescence-activated cell sorting (FACS) analysis were purchased from BD Biosciences (San Jose, CA, USA) and ThermoFisher Scientific (Waltham, MA, USA).

Techniques: Isolation, Flow Cytometry, Marker, Standard Deviation