70 μm nylon mesh

Femurs were extracted from 6- to 8-week-old C57BL/6J mice (University of Maryland, Baltimore [UMB], Veterinary Resources Breeding Facility) and flushed with Dulbecco's minimal essential medium (DMEM) supplemented with 10% low-endotoxin FBS (Gemini Bioproducts, West Sacramento, CA), 30% L929 cell supernatants (as a source of colony stimulating factor 1 [CSF-1]), 1% nonessential amino acids (ThermoFisher Scientific, Waltham, MA), 1% HEPES (ThermoFisher Scientific, Waltham, MA), and 1% penicillin-streptomycin (ThermoFisher Scientific, Waltham, MA). Bone marrow cells were passed through a 70-μm nylon mesh (ThermoFisher Scientific, Waltham, MA) to remove debris and placed into a T175 flask (Costar, Corning, NY) for culture and differentiation. Fresh medium was added on the day after extraction and every other day subsequently for at least 1 week. The day before an assay, cells were scraped, spun down at 1,100 rpm (125 × g) for 10 min, and resuspended at a concentration of 5 × 10⁵ cells per ml in medium lacking penicillin-streptomycin. Cells were plated at a concentration of 5 × 10⁵ cells per well in a 24-well plate (Costar, Corning, NY).

Mice were sacrificed by CO₂ exposure and the following tissues collected for flow cytometry analysis: blood was collected via cardiac puncture, followed by immediate lysis of red blood cells; spleens were rapidly dissected into RPMI with 10% FBS, and the bone marrow was collected by flush using a 23G needle (BD Biosciences, Franklin Lakes, NJ). Single cell suspensions were made by pressing the tissues through a 70 μm nylon mesh (ThermoFisher). Cellular composition and expression of GFP was analyzed after staining with antibodies specific for CD3, CD4, CD8, CD19, Thy1.2, CD11b, Ly6C and Ly6G. All antibodies used were obtained from eBioscience (San Diego, CA). Flow cytometry was performed using FACSCanto II (BD Biosciences) and the results were analyzed with the FlowJo software (TreeStar Inc., Ashland, OR).

PGECs were isolated from both male and female mice (P7) by a method described previously^{44 (link),45 (link)} with some modifications. Briefly, Sidt1^+/+ or Sidt1^−/− mouse pups (P7) were sacrificed after fasting for 12 h, the gastric epithelial tissues were separated from the stomach, divided into 1–2 mm pieces and digested with 0.125% trypsin-EDTA (Gibco) for 5 h at 4 °C and 20–25 min at 37 °C continuously, followed by termination with fetal bovine serum (FBS; Gibco). The digested tissue pieces were filtered through a 70-μm-nylon mesh (Thermo Fisher Scientific), and then centrifuged at 400× g for 3 min. The harvested cells were plated on PDL-coated cell culture dishes with M199 medium (Hyclone, SH30253.02) supplied with 10% FBS, 1% penicillin-streptomycin (Gibco), 100 mg/L heparin (MCE, HY-17567), 1× ECGS (ScienCell^TM, Cat# 1052) and 10 μg/L EGF (Novoprotein, C026). After 2 days of culture, the primary cells were used in the following experiments.

Mouse heart cells flow cytometry analysis were processed according to the method described by Chen et al [31 (link)]. The mice were anesthetized with 50 mg/kg pentobarbital. Heart tissues were cut into pieces and washed by cold PBS for three times to remove the blood cells. Then the tissues were digested by the enzyme (Liberase TH Research Grade) at 37 °C for 15 min. The supernatant containing the cells was transferred to the medium with 10% fetal calf serum to terminate the digestion. Residual tissues were digested with the enzyme at 37 °C for 15 min for further digestion. The digestion was repeated 3 to 4 times to isolate all cells from the heart tissues. All cell suspensions were collected and centrifuged at 250×g for 5 min. The supernatant was discarded, and the precipitate was resuspended in PBS and cells were filtered through a 70 μm nylon mesh (ThermoFisher, USA). Erythrocytes were removed by RBC lysis Buffer (ThermoFisher, USA). Cells were stained by 7-AAD, antibodies specific to Cd45-FITC and Ly6g-APC-Cy7 for 30 min. The cells were washed twice by cold PBS and subjected to flow cytometry (BD FACSCalibur) measurement. Data were analysed by FlowJo. 7-AAD negative cells were regarded as the alive cells. Leukocyte cell ratio was calculated by Cd45 positive cells/all alive cells. Neutrophil ratio was calculated by Ly6g positive cells/Cd45 positive cells.

Kidneys from mice of different conditions and genotypes were aseptically collected in ice-cold 1x sterile PBS. Using gentleMACS dissociation (Miltenyi Biotec), single-cell suspensions were digested in Accumax enzymatic solution (Cat# AM105; Innovative Cell Technology) as previously described. Cells were filtered and supplemented with 1xDMEM before centrifugation for 10 min at 300g. Red blood cell lysis buffer (Cat# 420301; BioLegend) was applied to resuspend cells and incubated for 5 min on ice. After lysis, the cell suspension was supplemented with DMEM and subjected to filtering through a 70-μm nylon mesh (Cat# 22-363-548; Thermo Fisher Scientific). Cells were washed again and resuspended in EasySep buffer (Cat# 20144; Stem Cell Technologies) for subsequent annexin V separation using EasySep Dead Cell Removal Kit (Cat# 17899; Stem Cell Technologies). To isolate kidney-derived leukocytes, anti-mouse CD45 microbeads (Cat# 130-052-301; Miltenyi Biotec) were applied and incubated, and CD45⁺ cells were flushed out with 1 ml of magnetic-activated cell sorting buffer (PBS containing 0.5% BSA and 2 mM EDTA). The process was repeated using anti-mouse CD117 microbeads (Cat# 130-097-146) on CD45⁻ cell suspensions to flush out ICs (CD45⁻/CD117⁺). Non-ICs were recovered from CD117⁻ cells (9 (link), 59 (link)).