Erythrocyte lysis buffer el

Pancreatic islets were isolated by collagenase digestion (0.7 mg/ml) (Sigma-Aldrich Chemie GmbH) and discontinuous Ficoll density gradient. Single-cell suspensions of pancreatic islets and lymphoid tissues were prepared using 70 μm cell strainers (Becton Dickinson, San Diego, CA, USA) and Hank’s buffer [1 x HBSS, 5% (v/v) FCS, 10mM HEPES; all Invitrogen]. Single cell suspensions from spleen were additionally subjected to red blood cell lysis (erythrocyte lysis buffer EL, Qiagen). Peripheral blood mononuclear cells (PBMCs) were obtained by retro-orbital sinus puncture [PBS supplemented with 10% (v/v) Heparin (Biochrom AG, Berlin, Germany)] and Ficoll (VWR, Darmstadt, Germany) gradient centrifugation. mAbs to CD3 (145-2C11), CD4 (RM4-5, GK1.5), CD8 (53–6.7), CD25 (PC61, 7D4), CD44 (IM7), CD62L (MEL-14), Vβ-4 (KT4), and CD49b (R1-2) were purchased from eBioscience (Frankfurt, Germany) or BD (Heidelberg). The samples were analyzed using a LSRII or sorted on a FACS Aria (all BD). Data were analyzed with the FlowJo software (Tree Star).

Infection of human blood ex vivo was performed as previously described [17 (link)] using FITC-labeled bacteria. Briefly, indicated concentrations of FITC-labeled bacteria in 100 μL of DPBS were combined with 1 mL of freshly drawn human blood containing heparin (Fresenius Kabi; 20 USP units/mL) in 2.0-mL microcentrifuge tubes and incubated at 37°C with end-over-end rotation. Preliminary experiments in which sodium citrate was used as an anticoagulant yielded results similar to those with heparinized blood. At indicated times, red blood cells were lysed using erythrocyte lysis buffer EL (Qiagen) following the manufacturer’s protocol (QIAamp RNA Blood Mini-Handbook), and leukocytes were resuspended in 200 μL ice cold DPBS. Pelleted cells were divided into 50-μL aliquots, stained with APC Mouse Anti-Human CD3, APC Mouse Anti-Human CD14, APC Mouse Anti-Human CD19, or PE Mouse Anti-Human CD66c (BD Biosciences), and analyzed using flow cytometry (FACSCalibur, BD Biosciences). Forward and side-scatter were used to set gates on leukocyte populations (lymphocytes, monocytes, and granulocytes). B cells were defined as CD19⁺ leukocytes, T cells as CD3⁺ leukocytes, monocytes as CD14⁺ leukocytes, and PMNs as leukocytes expressing high levels of surface CD66 (CD66^High).

Decidual tissue was processed as described in Kwan et al. (27 (link)). Briefly, decidua was washed and minced finely and flushed with HBSS^+/+ 2 to 3 times to ensure maximal release of leukocytes. Decidual cells were collected via filtration through 100 and 70 μm sieves and centrifugation (700 g for 10 min at 4°C). Isolated cells were suspended in RPMI-1640 + 10% FBS and fibroblasts eliminated through differential attachment to tissue culture plates (37°C for 20 min). Following incubation, remaining cells were passed through a 40 μm filter and incubated in erythrocyte lysis buffer EL (Qiagen, Toronto, ON, Canada) for 20 min at 4°C. The final cell suspension was incubated in serum-free protein block (Dako, Burlington, ON, Canada) for 1 h on ice and diluted to a final concentration of 10⁶cells/mL.