Pe cy5 conjugated anti cd4

FITC-conjugated anti-CD3, APC-conjugated anti-CD4, APC-conjugated anti-CD8, and their corresponding isotype controls were purchased from eBioscience (San Diego, CA). PE-Cy5-conjugated anti-CD4, PE-Cy5-conjugated anti-CD8, FITC-conjugated anti-CD44, and their corresponding isotype controls, were obtained from BioLegend (San Diego, CA). PE-conjugated anti-CD45RB, PE-conjugated anti-Fas, and their corresponding isotype controls were purchased from BD Bioscience (San Jose, CA). FITC-conjugated anti-CD28 and its corresponding isotype control were purchased from Serotec (Oxfordshire, UK). Rabbit polyclonal primary anti-Ki-67 and its isotype control were purchased from Abcam (Cambridge, UK) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. PerCP-conjugated donkey anti-rabbit IgG antibody was obtained from Santa Cruz Biotechnology. T cell mitogen concanavalin A (Con A) was obtained from Sigma (St. Louis, MO).

Spleens of the control group or LPS-challenged apoM^+/+ and apoM^−/− mice were harvested aseptically and filtered through cell strainers to produce a single-cell suspension. After lysis of red blood cells, the spleen suspensions were washed three times with PBS and adjusted to a concentration of 1.0 × 10⁷/mL. To detect the changes in lymphocyte subgroups, the prepared splenocytes (1.0 × 10⁷/mL, 100 μL) were incubated with 0.5 μL each of fluorescently labeled antibodies for 30 minutes at 4°C protected from light and washed three times with PBS before analysis. Fluorescently labeled antibodies included FITC-conjugated anti-CD3, PE-Cy5 conjugated anti-CD4, and PE-Cy7 conjugated anti-CD8 (BioLegend, San Diego, CA). The stained cells were then analyzed by flow cytometry immediately (FC500, Beckman, USA).