Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called “organoids,” to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems.

No abstract

We compared crural diaphragm electrical activity (EAdi) with transdiaphragmatic pressure (Pdi) during varying levels of pressure support ventilation (PS) in 13 intubated patients. With changing PS, we found no evidence for changes in neuromechanical coupling of the diaphragm. From lowest to highest PS (2 cm H(2)O +/- 4 to 20 cm H(2)O +/- 7), tidal volume increased from 430 ml +/- 180 to 527 ml +/- 180 (p < 0.001). The inspiratory volume calculated during the period when EAdi increased to its peak did not change from 276 +/- 147 to 277 +/- 162 ml, p = 0.976. Respiratory rate decreased from 23.9 (+/- 7) to 21.3 (+/- 7) breaths/min (p = 0.015). EAdi and Pdi decreased proportionally by adding PS (r = 0.84 and r = 0.90, for mean and peak values, respectively). Mean and peak EAdi decreased (p < 0.001) by 33 +/- 21% (mean +/- SD) and 37 +/- 23% with the addition of 10 cm H(2)O of PS, similar to the decrease in the mean and peak Pdi (p < 0.001) observed (34 +/- 36 and 35 +/- 23%). We also found that ventilator assist continued during the diaphragm deactivation period, a phenomenon that was further exaggerated at higher PS levels. We conclude that EAdi is a valid measurement of neural drive to the diaphragm in acute respiratory failure.