Miltenyi macsquant analyzer 10

Single‐cell suspensions of placental leukocytes were prepared, as previously described (Shields et al. 2018). Briefly, one placenta from each rat was homogenized and filtered through a 70‐μm cell strainer and resuspended in 15 mL of Rosswell Park Memorial Institute medium (RPMI) (10% FBS). Whole blood was collected in an EDTA tube and diluted with 5 mL of RPMI. Peripheral blood mononuclear cells (PBMCs) and placental lymphocytes were isolated by centrifugation on a cushion of Ficoll‐Isopaque (Lymphoprep, Accurate Chemical & Scientific Corp., Westbury, NY) according to the instructions of the manufacturer. Single‐cell suspensions (1 × 10⁶ cells) were stained for flow cytometry after blocking with 10% goat and mouse serum. Antibodies used for flow cytometry were as follows: VioGreen anti‐CD3 (Miltenyi Biotec, Auburn, CA), anti‐ANK61 antibody (Abcam, ab36392), antimouse IgG FITC (Abcam, ab97239), anti‐ANK44 (Abcamab36388), and antimouse IgG AlexaFluor 405 (Abcam, ab175663). Flow cytometry was performed on the Miltenyi MACSQuant Analyzer 10 (Miltenyi) and analyzed using FlowLogic software (Innovai, Sydney, Australia). Lymphocytes were gated in the forward and side scatter plots. After doublet exclusion, additional gates were set using fluorescence minus one (FMO) controls. Results are expressed as % of cells in the gated lymphocyte population.