Ebioscience intracellular fixation permeabilization buffer

Intestinal endothelial cells from mT/mG pups were purified as previously described^{50 (link)}. Myeloid cells were purified from either Cx3cr1-GFP or wild-type (WT) pup intestinal tissues. All cell isolations were performed by pooling 8–15 pup intestines in each group to yield enough cells for experiments. In all, 4 × 10⁴ endothelial cells and 2 × 10⁴ myeloid cells or macrophages were cultured on Matrigel separately or together, in endothelial culture media without or with IGF-1 (100 ng/ml) and/or PPP (500 nM). Cell sprouting images were taken by a Zeiss 880 Confocal Laser Scanning Microscope at 10x magnification. The length of the sprouts from each image was measured using photoshop. Alternatively, isolated macrophages and endothelial cells were cultured in vitro either separately or together at a 1:2 ratio in triplicates in 24-well plates. Cells were collected after 48 h of culture and stained with indicated markers or isotype control antibodies for flow cytometric analysis after gating on live singlet cells. IGF-1 was stained after cell fixation and permeabilization using eBioscience™ Intracellular Fixation & Permeabilization Buffer (88-8824, Thermo Fisher) post cell surface marker staining. Endothelial cells were identified as Tie-2⁺CX3CR1⁻ population since the CD31 marker could not be used, being no longer detectable after cells have been cultured in vitro and retrieved.

Cells were stained with APC-anti-mouse CD3e (553066; BD Pharmingen, San Diego, CA) and PE-anti-mouse CD4 (557308; BD Pharmingen) for 30 min at 4 °C, followed by washing twice with PBS containing 1% FBS and 0.03% sodium azide. For intracellular staining, cells were fixed and permeabilized using eBioscience™ Intracellular Fixation & Permeabilization Buffer (Thermo Fisher Scientific) followed by staining with FITC-anti-mouse IFN-γ (554411; BD Pharmingen). After washing twice with PBS containing 1% FBS and 0.03% sodium azide, the cells were analyzed by FACScalibur (Becton, Dickinson and Company, Franklin Lakes, NJ) using FlowJo software (Becton Dickinson).

Lungs were cut into fine pieces (1 mm³) in RPMI1640 containing 2.5 mg/ml collagenase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 1 mg/ml of dispase II (Roche, Basel Switzerland), and 0.02 mg/ml of DNase I (Millipore, Barrington, USA). The cell suspension was incubated at 37°C for 21 min with pipetting every 7 min and passed through a 70-µm cell strainer. After washing, red blood cells were lysed and Fc receptors were blocked. Cells were stained with fluorescence-conjugated monoclonal antibodies for surface molecules shown in Supplementary Table 3 and incubated on ice for 15 min. After washing, cells were ready for the flow cytometric analysis of surface molecules. Regarding staining with prosurfactant protein C (proSP-C), cells were further fixed and permeabilized with eBioscience Intracellular Fixation/Permeabilization buffer (Thermo Fisher Scientific, Waltham, USA), and then stained with anti-proSP-C polyclonal Ab (pAb) (Abcam, Cambridge, UK) followed by staining with Alexa Fluor 555-conjugated anti-rabbit pAb (Abcam). The flow cytometric analysis was performed using BD LSRFortessa TM (BD Biosciences, San Jose, USA), and data analyses were conducted using FlowJo software ver. 10.7.1 (BD Biosciences). BD FACS Aria™ III (BD Biosciences) was used for flow cytometric sorting.