Collagenase type 4

To further investigate the effect of elevated LPS concentrations during SARA on the endometrial epithelium TJs, the sheep uterus of the LC group was collected and primary endometrial epithelial cells were cultured [55 (link)]. Antibiotics (50 IU/mL penicillin and 50 IU/mL streptomycin; Solarbio) at 37 °C in PBS and 75% alcohol were used to sequentially wash the endometrial tissue thrice, and the endometrial epithelium was cut with 0.2% collagenase type 4 (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 10 min. Centrifugation was performed at 1200 rpm for 5 min to remove the supernatant, followed by washing with PBS thrice. The cells were then cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 complete medium suspension containing 100 IU/mL penicillin (Solarbio), 100 IU/mL streptomycin (Solarbio), and 10% fetal bovine serum at 37 °C with 95% O₂ and 5% CO₂ in a humidified incubator. The medium was changed every 2 d, and the cells were split upon reaching 80–90% confluency and cultured in six-well plates (Corning, Glendale, AR, USA), which contained 2 mL of medium. During treatment, DMEM/F12 was added to the original culture for 12 h and was used to treat according to the test needs for 24 h, after which the supernatant was discarded, and the cells were washed with PBS precooled at 4 °C three times and stored at −80 °C until use.

Primary hepatocytes were isolated from 6–8-week-old C57BL/6 mice (Charles River Laboratories, Wilmington, MA, USA) as described previously^{27 (link)}. Briefly, after the abdomen was opened, the intestines were displaced to the left, and the portal vein was exposed. The chest was opened, and the hepatic vein was cannulated in a retrograde fashion via an incision in the right atrium with a 24-gauge needle connected to a variable-speed pump that delivered perfusion solution 1 (142 mM NaCl, 6.7 mM KCl, and 10 mM HEPES) at a rate of 8 mL/min. The hepatic portal vein was cut to allow the perfusion fluid to escape. After 2 min, perfusion solution 1 was switched to perfusion solution 2 (66.7 mM NaCl, 6.7 mM KCl, 100 mM HEPES, and 4.8 mM CaCl₂·2H₂O) containing 25 mg of collagenase type 4 (Sigma) and 2% bovine serum albumin (BSA) in 50 mL, and the perfusion was continued for another 2–3 min at the same rate. Perfused liver was then excised and transferred to a 60-mm culture dish containing the primary cell culture medium. The liver was broken apart gently with forceps. Cells were counted and plated in medium 199 (Sigma) supplemented with 10% fetal bovine serum (FBS), 1 U/mL penicillin, 1 µg/mL streptomycin, 10 nM dexamethasone, and 23 mM HEPES. The cells were incubated at 37 °C in a 5% carbon dioxide (CO₂) atmosphere.

Blood was collected weekly to assess T cell phenotype and absolute count. On day 42 prior to organ harvest, mice were intravenously injected with 1.5µg of anti-CD4-BV650 and anti-CD8a-BV650 antibody 2 minutes before euthanasia to label circulating blood cells. The spleen, axillary lymph nodes, bone marrow, kidneys, liver, lungs, and blood were then procured. Kidney and lung samples were chopped and digested for 30 minutes at 37˚C with 2mg/ml Collagenase (type 4, Sigma-Aldrich) and 50µg/ml DNAse (ThermoFisher) in HBSS. Digested lungs were then homogenized, filtered, and washed in FACS buffer (PBS with 2% FBS). Livers were homogenized manually, filtered through 40μm strainers, and spun lightly at 300rpm to pellet the hepatocytes. The liver supernatant and digested kidneys were resuspended in a 40% Percoll solution, overlaid on 70% Percoll, and spun at 2000rpm for 20 minutes with the brake off. The buffy coats were isolated and washed in FACS buffer. Bone marrow from tibia and fibula were flushed with a 21-gauge needle using PBS and homogenized into single cell suspensions. Spleens and lymph nodes were processed into single cell suspensions, and blood, spleen, and bone marrow were lysed with Fixative-Free Lysing Solution following manufacturer’s instructions (Invitrogen). Each tissue was then washed in FACS buffer and stained with antibodies for flow cytometry.

Mice were sacrificed by CO₂ asphyxiation and tissues removed aseptically. Mice were injected i.v 3 min prior to euthanasia with anti-CD45-APCCy7 (5 μg; BD Biosciences, CA) to distinguish labeled vascular leukocytes from unlabeled lung parenchymal cells (29 (link)). BAL were obtained by inflating the lungs with 1 ml PBS and collecting the fluid to isolate the cells. The post-caval lung lobe was collected into 10% neutral buffered formalin, paraffin embedded and processed for hematoxylin and eosin staining using standard techniques. For isolation of lung leukocytes, lung tissue in complete RPMI media [L-glutamine and 25 mM Hepes (Invitrogen, CA), FCS (10% v/v), 2-mercaptoethanol (50 μM; Sigma, MO) and PenStrep (100 U/ml; Invitrogen)] was digested with collagenase type 4 (50 U/ml; Sigma) and DNAse I (13 μg/ml; Sigma) at 37°C for 45 min prior to homogenization and multiple filtration steps. Lymph nodes and spleens were filtered (70 μm) in complete RPMI and the leukocytes pelleted by centrifugation (500 g). Erythrocytes were removed by ACK lysis buffer.