Journal: Journal of immunological methods

Article Title: In vitro analysis of antigen induced T cell-monocyte conjugates by imaging flow cytometry

doi: 10.1016/j.jim.2018.06.014

Figure Lengend Snippet: Antigen induced lymphocyte proliferation and IFN-γ production in vitro: PBMC from healthy blood donors (n = 6) labeled with CellTrace Violet (CTV) were cultured for 6 days in the presence of PPD, ds-Red BCG, anti-CD3/CD28 or media alone (UNS). Cells were harvested at various time points, fluorochrome stained and analyzed by flow cytometer for CTV dilution. (A) Representative histogram overlays; 0, 4, 5 and 6 days in vitro are shown here with the gate indicating CTVLow CD4+ events. (B) Frequency of CD4+ T cells with CTV dye dilution (CTVLow events) in response to anti-CD3/CD28 (dotted line with triangles), PPD (dashed line with circles), ds-Red BCG (solid line with squares), or UNS (gray line with diamond) at days 0, 4, 5 & 6. Error bars show mean ± SD. (C) Representative dot plots of IFN-γ secreting CD4+ T cell. (D) Proportion of IFN-γ secreting CD4+ T cells in response to PPD (circles), ds-Red BCG (squares), anti-CD3/CD28 (inverted triangle) or UNS (triangles). Horizontal bars indicate median values.

Article Snippet: Antibodies used include anti-CD3 (clone OKT3), anti-CD28 (clone CD28.2, Cat# 555725, BD Pharmingen), anti-CD3-FITC (clone UCHT1, Cat# 21620033, Immunotools), anti-CD14-APC (clone MEM-15, Cat# 21279146, Immunotools), anti-IFN-γ PerCP Cy-5.5 (Clone B27, Cat # 560704, BD biosciences), rabbit polyclonal IgG (C-20) anti-human NF-κB primary antibody (SC-372R, Santa Cruz Biotechnology), Alexa Fluor 594, goat anti-rabbit IgG (H + L) secondary antibody (Cat# {"type":"entrez-nucleotide","attrs":{"text":"R37117","term_id":"794573"}} R37117 , Molecular Probes, Invitrogen).

Techniques: In Vitro, Labeling, Cell Culture, Staining, Flow Cytometry