Facs canto flow cytometer

CD4⁺ CD25^- T and Treg cells stimulated with rhMGL-Fc, were cultured alone or co-cultured (Tregs:CD4⁺ CD25^- T cells, 1:5) with anti-CD3 (1 μg/ml, OKT3) (eBioscience) and anti-CD28 (1 μg/ml)(BioLegend) antibodies for 4 days at 37°C. CD4⁺ CD25^- T cells was pre-treated with CarboxyFluorescein Succinimidyl Ester (1 μM, CFSE) (Life Technologies) and cell proliferation was monitored through progressive halving of fluorescence using FACSCanto flow cytometer. All experiments were performed in triplicate wells. One hundred percent proliferation was defined as the proliferation of CD4⁺ CD25^- T cells alone.

The total number of the CD45⁺ cells, present in the sdLN and in the skin, was calculated using absolute counting beads (CountBright, Life technologies, Eugene, OR, United States) in the FACS Canto Flow cytometer.
The cells that were stained for extracellular markers or intracellular cytokines were analyzed using a FACS Canto Flow cytometer (BD Biosciences, San Jose, CA, United States). The results were analyzed using FlowJo (Tree Star, Ashland, OR, United States) software.

C57BL/6 mice were i.d. injected in the ear with iC (5 × 10⁵ iC) plus the CT (1 μg). After 31 days, ear thickness was measured and mice were intraperitoneally (i.p.) inoculated with anti-CD4 monoclonal antibody (clone GK 1.5) (250 μg) or irrelevant antibody (Rat IgG1 κ Iso Control, Clone eBRG1, eBioscience) (250 μg). After 24 h of inoculation with the anti-CD4 or irrelevant antibody, ear thickness was measured and mice were challenged with an i.d. injection in the ears with iC (5 × 10⁵ iC). Ear thickness was measured 24 and 48 h post-challenge. The sdLN cells and ear skin cells were obtained, stained for CD45, CD4, TCR-β, CD44, CD62L, CCR7, and CD69 markers, and analyzed by flow cytometry.
The total number of the CD45⁺ cells, present in the sdLN and in the skin, was calculated using absolute counting beads (CountBright, Life technologies, Eugene, OR, United States) in the FACS Canto Flow cytometer.