Cell culture trypsin egta

Mice were euthanized under isoflurane anesthesia, and the CP from all four ventricles of six mice were dissected and collected in 4 °C HBS (in mM: 145.0 Na⁺, 3.6 K⁺, 1.8 Ca²⁺, 0.8 Mg²⁺, 138.6 Cl⁻, 0.8 SO₄²⁻, 5.5 Glucose, 10.0 HEPES, 2.0 PO₄²⁻, with pH 7.4). The pooled CP tissues were incubated in 50 µg/mL Concanavalin A fluorescein (Vector Laboratories, Oxfordshire, GB) in HBS for 10 min at 37 °C, washed and digested in 2 μg/mL dispase (Invitrogen, Carlsbad, CA, USA) and 2 μg/mL collagenase B (Roche, Basel, Switzerland) in calcium-free HBS for 30 min at 37 °C, and incubated in 1:1 mixture of TrypLE Select Enzyme (Thermo-Fisher, Waltham, MA, USA) and cell culture trypsin/EGTA (Thermo-Fisher) supplemented with 1 mg/mL DNase (Sigma, St. Louis, MA, USA) for 10 min at 37 °C. The preparation was inspected on the microscope, passed through a 50 µm filter, and incubated with propidium iodide added before FACS for exclusion of dead cells. Cells were sorted into fluorescein positive and fluorescein negative samples by 4-way purity sorting on a FACSAria III (BD Biosciences, San Jose, CA, USA). The yield and quality control for the FACS isolation of the used CPECs was reported previously [6 (link)].

Mice were anesthetised by isoflurane inhalation and euthanized by cervical dislocation. The choroid plexi (CP) from all brain ventricles were removed and placed in HEPES‐buffered saline (HBS) on ice (Table 1). Pooled CP tissues from 6 to 8 mice were incubated in 50 μg/mL concanavalin A fluorescein (Vector Laboratories) in HBS for 10 min at 37°C, digested in 4 μg/mL dispase (Invitrogen) and 4 μg/mL collagenase B (Roche) in calcium‐free HBS for 30 min at 37°C, and incubated in a 1:1 mixture of TrypLE Select Enzyme (Thermo‐Fisher) and cell culture trypsin/EGTA (Thermo‐Fisher) supplemented with 1 mg/mL DNase (Sigma) for 10 min at 37°C. The preparation was passed through a 50‐μm filter, and propidium iodide was added before Fluorescence‐activated cell sorting (FACS) for exclusion of dead cells. Cells were sorted into fluorescein‐positive and fluorescein‐negative samples by 4‐way purity sorting on a FACS Aria III (BD Biosciences). The cells were sorted using a 70‐μm nozzle, at 70 psi and 12–20 kHz. After FACS, the samples as well as control CP samples were spun for 1 min in a table‐top micro‐centrifuge and the pellets were processed for total RNA isolation. Sample purity was validated by RT‐PCR with primers targeting the epithelial marker Claudin‐1 (Cldn1) and the endothelial marker Claudin‐5 (Cldn5).