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Total RNA was isolated from whole hearts, kidneys, livers, lungs, and spleens as described elsewhere.14 Female mice were anesthetized with 200 mg/kg ketamine (Ketalean, 100 mg/mL; Bimeda-MTC Animal Health Inc., Cambridge, ON, Canada) and 10 mg/kg xylazine hydrochloride (Rompun, 20 mg/mL; Bayer Inc., Toronto, ON, Canada) by i.p. injection. The mice were positioned in dorsal recumbency, and the chest/abdomen surfaces were shaved and prepared with 70% ethanol. A ventral midline incision (approximately 1 cm) was made to allow exteriorization of the cecum. The cecum was ligated 1 cm from the apex with 3�C0 silk sutures and penetrated through and through using a 22-gauge needle. The abdominal incision was then closed in two layers with 4�C0 nylon sutures. Sham surgery, LY2109761 price in which the cecum was exteriorized and manipulated as described but not ligated or punctured, was performed in control animals. Immediately after surgery, the animals received fluid resuscitation with 50 mL/kg saline injected s.c. Saline or MSCs were injected 6 hours after the animals had undergone CLP to induce sepsis. Saline or cultured male MSCs (at 2.5 �� 105 cells, 100 ��L total volume) were slowly infused via a cannula inserted into the jugular vein 6 hours after sham operation or CLP. The mice were randomized to receive saline or MSCs. After infusion, the cannula was withdrawn, the vein was ligated, and the mice were returned to the vivarium and had free access to food and MAPK water. The mice were sacrificed 28 hours after the initial sham or CLP procedure to collect tissue samples for analysis. The heart, liver, spleen, learn more lungs, and kidneys were snap frozen and stored at ?80��C for microarray analysis. Total RNA from whole spleens, lungs, livers, hearts, and kidneys (collected 28 hours after CLP) from five animals per group (sham/saline, CLP/saline, and CLP/MSCs) was extracted using TRIzol reagent (Invitrogen, Burlington, ON, Canada) and purified using RNeasy (Qiagen Inc., Chatsworth, CA) per manufacturer specifications. RNA quality was ensured by spectrophotometric analysis (OD260/280) and gel visualization. Animals intended for microarray analysis were preselected on the basis of clear group assignment based on physiologic parameters of inflammation (P
