Anti allophycocyanin microbeads

Cells for transfer were obtained from either the myometrium or the spleen of pregnant mice preadministered α‐GalCer on Gd 7.5. CD1d tetramer⁺ iNKT cells were isolated by positive selection with anti‐allophycocyanin MicroBeads (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions. α‐GalCer‐induced CD1d tetramer⁺ iNKT cells obtained from either the myometrium or the spleen of pregnant mice were i.v. transferred into other pregnant mice on Gd 7.5 (3 × 10⁴ cells for each mouse). Three days later (Gd 10.5), the fetal loss rate was analyzed.

As indicated above, bone marrow cells were harvested from the femurs of virgin female B6 mice and were resuspended, and red blood cells were eliminated. The obtained bone marrow cells (8 × 10⁶) were cultured in 4 mL of RPMI 1640 with 10 ng/mL IL‐4 and 10 ng/mL GM‐CSF in six‐well flat‐bottom multiwell plates (Corning) at 37°C. The culture medium was replaced with fresh medium, and on day 3, the floating cells were removed, and 5 μg/mL α‐GalCer was added. On day 6, floating and loosely adherent cells were collected and washed. DEC‐205⁺ DCs or 33D1⁺ DCs were isolated by positive selection with anti‐allophycocyanin MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. To obtain purified splenic T cells, spleen cells were passed through a nylon wool column as previously described 29 and the purified splenic T cells were placed into a 96‐well U–bottom multiwell plate (Corning). Then, bone marrow‐derived DCs (2 × 10⁴) were added to the splenic T cells (1 × 10⁵) with 5 μg/mL α‐GalCer and 20 μL of T‐STIM (BD Biosciences). Five days later, the floating cells were collected, washed, and analyzed using a FACSCantoII flow cytometer.