Human PBMCs and SGECs were cocultured at the ratio of 1: 1 for 3 days in the presence of 2.5 [micro]g/ml phytohemagglutinin
with or without EVs derived from BM-MSCs or iPSC-MSCs (5 [micro]g/ml) and then examined for gene expressions in isolated PBMCs (a) and SGECs (b) by qRT-PCR.
(10 [micro]g/mL) was used as a positive control.
Nitrotetrazolium blue tetrazolium (NBT), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), phytohemagglutinin
(PHA), dioxin, and phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St.
CFSE-stained PBMCs (5 x [10.sup.5]; n=6) were cultured in RPMI-1640 containing 10% FCS (Gibco, Invitrogen Corporation), 2 mM L-glutamine, antibiotic-antimycotic cocktail (Sigma-Aldrich Corporation), either with or without phytohemagglutinin
(PHA) (1 [micro]g/mL).
Criteria: The positive result: (1) The negative control hole spot points were 0–5, and (A antigen or B antigen spot points) – (the negative control hole spot points) ≥6; (2) while the negative control hole spots ≥6, and (A antigen or antigen B spot points) ≥2 times (the negative control hole spots); the negative result does not meet the positive criteria, and the control hole of aseptic phytohemagglutinin
Ltd., Saint Louis, Missouri) or 1.2 [micro]g/mL of phytohemagglutinin
(PHA; BiochromAG, Berlin), in the presence or absence of two BS extracts (0.1 [micro]g/mL).
(PHA), which triggers inflammation in macrophages, was used as a positive control.
(used at doses of 5 [micro]g/mL) and concanavalin A (used at doses of 5 [micro]g/mL) were purchased from Sigma-Aldrich (St.
PBMCs were cultivated without stimulus, stimulated with soluble leishmania antigen (SLA; 5 [micro]g/ml), in the presence or absence of DI4G (300 nM), and phytohemagglutinin
(PHA, 10 [micro]l/ml; Gibco BRL, Grand Island, NY) at 37[degrees]C in an atmosphere of 5% C[O.sub.2].
Finally, the most important follow-up testing is that of T cell proliferation in response to mitogens such as phytohemagglutinin
Cytogenetic study: Chromosomal analysis was performed after 72 hr phytohemagglutinin
(PHA) stimulation of peripheral blood lymphocytes culture based on standard methods.