Cd4 t cell isolation kits 2

Peripheral blood samples (60 to 100 mL) were obtained from CL patients from the southwestern region of Colombia (Departments of Valle del Cauca and Nariño). Participants included 8 males and 2 females of 20 to 65 years of age (median = 27), who had typical cutaneous lesions, were parasitologically diagnosed by smear, culture or biopsy, and had not received anti-leishmanial treatment before enrollment. Blood samples from four healthy subjects from non-endemic areas were obtained as controls. All subjects had negative serology for HIV and HTLV-1. All participants provided written informed consent. The study protocol, consent forms and all procedures were approved by the CIDEIM Institutional Review Board for the ethical conduct of research involving human subjects.
PBMCs were isolated by centrifugation over Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). B cells and CD4 T cells were isolated by MACS using the B cell isolation and CD4⁺ T cell isolation kits II, respectively (Miltenyi Biotec, Bergisch-Gladbach, Germany) following the manufacturer’s instructions. Purity assessed by staining with anti-CD20 or anti-CD4 was ≥95% for both populations. Ramos cells were obtained from the American Type Culture Collection (Rockville, MD).

The binding of ArtinM to CD3 receptor was determined on CD4⁺ T cells isolated from spleen cell suspensions using CD4⁺ T cell isolation kits II and MS columns from Miltenyi-Biotec according to the manufacturer’s instructions. In the first assay, the cells were fixed and incubated with ArtinM (25 μg/mL) for 40 min at 4 °C. After two washes with phosphate-buffered saline (PBS), the cells were incubated with an anti-CD3 PE-Cy5 antibody (10 μg/mL; clone 17A2; BD Biosciences) for 40 min at 4 °C. The percentage of labeled cells was determined using flow cytometry (Guava EasyCyte). A functional inhibition assay was carried out by pre-incubating CD4⁺ T cells with either the anti-CD3 antibody (5 μg/mL) or an anti-CD28 antibody (5 μg/mL; clone 37.51; BD Biosciences) for 40 min at 4 °C. After washing with RPMI medium, the cells were stimulated for 48 h with jArtinM (78 nM). The culture supernatants were tested by means of ELISA for IL-2.