Total RNA was isolated from the lungs using RNeasy mini kit according to the manufacturers instructions. To remove genomic DNA contamination, Palbociclib clinical trial isolated RNA samples were treated with 1 U DNase ��g RNA for 15 min at 37 C. One microgram of total RNA was used in a 20 ��l reaction to synthesize Inhibitors,Modulators,Libraries cDNA using Superscript H reverse transcriptase and oligo dTs as primers. Reverse transcription reaction was for 50 min at 42 C. Quantitative real time PCR was performed using the I Cycler IQ detection system in combination with the IQ SYBR Green Real Time PCR Supermix. The PCR conditions included initial denaturation in one cycle of 10 min at 95 C followed by 40 cycles of 20 s at 95 C, 20 s at 60 C, and 20 s at 72 Inhibitors,Modulators,Libraries C. The relative expressions were calculated as 2 x 1 mean control Inhibitors,Modulators,Libraries 2. where CT is calculated as CT CTGOI CTHKG.

Inhibitors,Modulators,Libraries Primer sequences are provided in the Additional file 1 Table S1. Regulation of CRLR and RAMP 1 3 mRNA in uninfected, spontaneously breathing and uninfected, mechanically ventilated groups has been published previously. Lung permeability Human serum albumin was intravenously injected 90 min prior to the end of the experiment. After ligation of the left stem bronchus bronchoalveolar lavage was performed twice with 400 ��l PBS each. From each BALF portion, 250 ��l were pooled and BAL HSA concentration, as well as plasma concentration of HSA was determined by ELISA. Permeability was assessed by calculating the cHSA BAL plasma ratio as described. Hypoxic vasoconstriction in precision cut lung slices PCLS were prepared as described previously.

Briefly, mice were killed by cervical dislocation and the airways were filled with 1. 5% low melting point agarose. After solidification of the agarose, the lungs were cut into 200 ��m thick slices. The agarose was removed by incubation of the PCLS in phenolred free minimal essential medium continuously gassed with 21% O2, 5% CO2, 74% N2 for at Inhibitors,Modulators,Libraries least 2 h at 37 C. To analyze vasoreactivity of individual cross sectioned intra acinar arteries, PCLS were transferred into a flow through superfusion chamber. At the beginning of each experiment the capability of the vessel to contract in response to the thromboxane analogue U46619 and to dilate after application of the NO donor sodium nitroprusside was checked. After washing out these drugs with normoxic gassed phenolred free MEM PCLS were incubated with hypoxic gassed medium.

After 15 min 500 nM of murine AM was added to the hypoxic medium. After a second wash out PCLS were again challenged with U46619 in presence of 500 nM AM when the hypoxic incubation was performed in presence of AM or solvent, respectively. Pictures of the artery were taken every 2 min using scientific assay an inverted microscope mounted on the superfusion chamber. Changes of the luminal area of the vessels were evaluated by manually lining the inner boundaries. The luminal area at the beginning of hypoxia was defined as 100% and vasoreactivity was expressed as relative decrease or increase of this area.