Fibrinolytic niche is requested for alveolar type 2 cell-mediated alveologenesis and injury repair

Ali Gibran; Runzhen Zhao; Mo Zhang; Krishan G. Jain; Jianjun Chang; Satoshi Komatsu; Xiaohui Fang; Beiyun Zhou; Jiurong Liang; Dianhua Jiang; Mistuo Ikebe; Michael A Matthay; Hong-Long Ji

freshly isolated AT2 cells suspended in DPBS + 10% FBS (2 ´ 10 5 cells/mL) were centrifuged at 600 rpm for 4 min by a Shandon CytoCentrifuge. Slides were air-dried overnight and stained using a modified PAP stain method2. Briefly, slides were stained with hematoxylin for 3.5 min. After rinsing in dH2O, slides were incubated in lithium carbonate for 2 min and rinsed with dH2O again. Slides were dehydrated in serial ethanol dilutions, then in xylene: ethanol (1:1) for 30 s, and finally in 100% xylene for 60 s. Slides were mounted with the Permount mounting medium. Randomly selected images were captured from 6 independent experiments with an Olympus BX41 microscope (40 ´). AT2 cells with large nuclei and blue colored granules spread in the cytoplasm were counted and calculated for purity (%). 2) Immunofluorescent stain: cytospanned freshly isolated AT2 cells and cells cultured on coverslips for 48 h were incubated with rabbit anti-proSP-C (1:500) overnight at 4 o C. Secondary antibody, either Alexa Flour 488 or 568-conjugated anti-rabbit IgG, was added to recognize proSP-C antibody. (Fig. S1A-C) . Cell viability was approximately 94%. Further, FACS showed that approximately 95% of cells were EpCAM positive (Fig. S1D-