Amnis flowsight imaging flow cytometer

The cellular mucosal immune system status of the broiler's intestinal tract, intraepithelial lymphocytes (CD3+ T lymphocytes, CD3+CD8+ cells, CD3+CD4+ cells, and CD3+CD4+CD8 cells) were measured. At post mortem, intraepithelial T lymphocytes were estimated from isolated jejunum of 40 birds (1 bird per cage) at 42 d of age according to the method defined by Röhe (2014) . The intestinal cell suspensions were stained with either a cocktail of T lymphocyte CD marker antibodies (CD3-AF647, CD4-FITC and CD8-PE; Southern Biotech, Birmingham, AL, USA) or a cocktail of isotype control antibodies (IgG1-PE, IgG1-FITC, and IgG1-AF647; Southern Biotech, Birmingham, AL, USA) for 30 min on ice in the dark according to the manufacturer's instructions.
Flow cytometric data were acquired on an Amnis FlowSight imaging flow cytometer (Millipore, Burlington, Massachusetts, USA). CD4-FITC and CD8-PE were detected using the 488 nm laser, and the CD3-AF647 was detected using the 633 nm laser. Signals from the isotype antibody cocktail were subtracted from the T lymphocyte CD antibody fluorescence. From the total cell population that was acquired, the CD3+ intact cell population was gated. Sub-gates were applied to the intact CD3+ cells population to determine the proportion of cytotoxic T lymphocytes (CD3+CD8+), T helper lymphocyte (CD3+CD4+) and double-stained T lymphocytes (CD4+CD8+).

BJ cells (10⁶ cells) were fixed using 4% solution of p-formaldehyde in PBS containing 0.01% Triton X-100 at room temperature for 10 min, washed using PBS and suspended in 70% ethanol at 4 °C for 20 min. To avoid unspecific binding, fixed cells were pre-incubated with 1% BSA in PBS for 20 min and then incubated with primary antibodies anti-p53 (1:200, MA5-12557) and anti-p21 (1:200, MA5-14949) at room temperature for 1 h and secondary antibody conjugated to Alexa Fluor Plus 488 (1:1000, A32723, A32731) (Thermo Fisher Scientific, Warsaw, Poland) at room temperature for 1 h. Fixed cells were then stained using 5 μg/ml PI solution for 10 min. Digital cell images were captured and intracellular localization of p21 and p53 was analyzed using flow imaging cytometry (Amnis® FlowSight® imaging flow cytometer and IDEAS software version 6.2.187.0, Merck Millipore, Warsaw, Poland).