The regulation of T cell homeostasis during pregnancy has important implications for maternal tolerance and immunity. support the idea that pregnancy is usually a state of dynamic T cell homeostasis and suggest that this state is partially supported by PD-1 signaling. and housekeeping genes were confirmed to Irsogladine be stable throughout gestation using the geNorm program (Vandesompele et al. 2002 The relative expression of PD-1 in each sample was then determined by dividing by the geometric mean of the housekeeping genes. Tissue preparation for flow cytometry Spleen and uterine draining lymph nodes (para-aortic and femoral) of pregnant mice and unmated controls were isolated in IMDM medium made up of 10% fetal bovine Irsogladine serum (Invitrogen) and 1μM beta-mercaptoethanol (Bio-Rad Laboratories) and single cell suspensions were generated by mechanical dissociation. The uteri of non-pregnant mice were removed by cutting at the cervix and ovaries and then uteri from 3-4 mice were pooled together. The uteri of pregnant mice were isolated by bisecting each uterine horn and peeling away fetal-placental units from the decidual attachment sites. Using a modification of a published methods (Tilburgs et al. 2006 uteri were cut into small pieces and enzymatically digested with 200 U/ml hyaluronidase (Sigma) 0.2 mg/ml DNAse I (Sigma) and 0.28 U/ml Liberase Blendzyme 3 (Roche Applied Science) in Hanks Balanced Salt Rabbit polyclonal to Caspase 10. Solution (Mediatech Inc.) containing 10% BSA (Sigma) for 20 minutes at 37°C. Samples were washed twice with PBS-0.1% BSA then pressed through 100μm mesh and exceeded through a MACS pre-separation filter (Miltenyi Biotec. Inc. Auburn CA USA) to remove cell clumps. In vivo BrdU assay T cell proliferation was decided using the previously described bromodeoxyuridine (BrdU) incorporation assay (Norton et al. 2009 Pregnant mice and unmated controls Irsogladine received four intraperitoneal injections of 1 1 mg BrdU (100ul of 10mg/ml BrdU in sterile PBS) (Sigma) in the 24 hours prior to euthanasia. One million spleen and uterine draining lymph node cells were treated with 0.5μM Fcγ Irsogladine III/II Receptor (BD Biosciences) and then stained with antibodies against CD4 CD8 and TCRβ in PBS-0.1% BSA for 30 min at 4°C. Samples were washed with sterile PBS (Mediatech Inc.) then fixed with PBS made up of 1% methanol-free formaldehyde (Ted Pella Inc. Redding CA USA) and permeabilized overnight in PBS-1% methanol-free formaldehyde made up of 0.01% Tween 20 (Sigma). The following day DNA was digested by treatment with 50U/ml of deoxyribonuclease I (Sigma) in buffer made up of 0.15M NaCl 4.2 MgCl2 (Sigma) at pH 5.0 for 15 min at 37°C. Cells were washed with PBS and stained with FITC-conjugated anti-BrdU for 30 minutes at 4°C. Samples were then washed with PBS-0.1%BSA and fixed with PBS-0.1% BSA-1% methanol-free formaldehyde. BrdU incorporation in TCRβ+CD4+ and TCRβ+CD8+ cells was detected by using a BD LSRII flow cytometer (BD Biosciences) and quantified using FlowJo software analysis (Tree Star Inc. Ashland OR USA). TUNEL assay to detect apoptosis The terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) flow cytometric assay was used to detect the nicked DNA in apoptotic cells as described previously (Norton et al. 2009 Briefly single cell suspensions of spleen and uterine draining node cells were treated with 0.5μM Fcγ III/II Receptor and stained with the same antibodies as described in the BrdU assay. Irsogladine Cells were fixed in PBS-1% methanol-free formaldehyde (Ted Pella Inc.) for 15 minutes washed with PBS (Mediatech Inc.) and then permeabilized by treatment with ice-cold 70% ethanol in PBS for 15 minutes. After washing with PBS cells were incubated with 10U of terminal deoxynuclotidyl transferase (TdT) and 6.25μM FITC-dUTP in 1X TdT reaction buffer with 2.5mM cobalt chloride (all from Roche Applied Science) for 1 hour at 37°C. Samples were then washed with PBS- 0.1% BSA and fixed with PBS-0.1% BSA-1% methanol-free formaldehyde. TUNEL positive TCRβ+CD4+ and TCRβ+CD8+ cells were detected by flow cytometry (BD LSRII BD Biosciences) and quantified with FlowJo software analysis (Tree Star Inc.). Mean Fluorescence intensity Single cell suspensions of spleen uterine draining node and uterus were treated with 0.5uM Fcγ III/II receptor (BD Biosciences) for 10 min to block non-specific antibody binding. Cells were then incubated with antibodies to CD4 CD8 TCRβ CD44 CD25 and PD-1 for 30 minutes at 4°C. Samples were washed with PBS-0.1% BSA and fixed.