The kidney microenvironment influences macrophage response and basement membrane homeostasis
The kidney microenvironment influences macrophage response and basement membrane homeostasis
Kidney leukocyte isolation - The kidney capsules were removed, and tissues minced, followed by digestion at 37°C for 30 minutes at 150 rpm in Hank's Balanced salt solution with calcium chloride and magnesium sulphate containing 0.1mg/ml Liberase TM Research Grade enzyme (05401127001, Roche) and 0.1mg/ml DNaseI (11284932001, Roche). Digested tissue was passed through a 40μm strainer and resuspended in Red Blood Cell (RBC) lysing buffer (Hybri-MaxTM) for 3 minutes at room temperature. Cells were enriched with CD45 microbeads (Miltenyi Biotec) and resuspended in PBS prior to flow staining and FACS.  Whole blood preparation - 500μl blood was collected into EDTA-coated syringes through cardiac puncture from mice. Suspensions were mixed with RBC lysing buffer for 3 minutes on ice twice and resuspended in PBS until flow staining.  FACS - Single cell suspensions (2.5 x 10^5 – 2.0 x 10^6 total cells) from the kidney and blood were washed with PBS. Subsequently, cells were stained with fluorochrome-conjugated extracellular targets (Table1c) for flow cytometry containing anti-CD16/CD32 (2.4G2, BD Biosciences) diluted in flow buffer containing PBS and 2% (v/v) FCS at 4°C for 15 minutes. Cells were washed with flow buffer and stained with 0.25μg/ml of DAPI before acquisition. Cells were sorted using the BD InfluxTM (BD Biosciences) into RLT buffer (QIAGEN) containing 0.1% (v/v) β-mercaptoethanol and stored at -80°C for RNA extraction. C57BL/6J (CD45.2) mice (Envigo, UK) were housed at The University of Manchester Animal Unit under SPF conditions. Male mice aged 10-12 weeks were used. Experiments were approved by The University of Manchester Local Review Committee and carried in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986. RNA was extracted from 1.5 – 2.0 x 10exp5 cells using the RNeasy micro kit (QIAGEN). Sample integrity was assessed using the RNA ScreenTape 2200 Tapestation assay and system (Agilent Technologies) and quantified using a Qubit 2.0 Fluorimeter (ThermoFisher Scientific). Libraries were prepared with the SMART-Seq v4 Kit (Takara Bio).