Llc pk1

The LLC-PK₁ (pig kidney cells) cell line was purchased from the ATCC. Cells were grown in Medium 199 with HEPES modification (Sigma) supplemented with 2.2 g/L NaHCO₃, 50 mg/L penicillin G, 50 mg/L streptomycin and 4% fetal calf serum (FCS). CCRF-CEM human lymphocytic leukemia cells were cultured in suspension (0.5 × 10⁶ to 1.0 × 10⁶ cells per mL) in RPMI 1640 medium (Sigma) supplemented with 10% FCS and the same antibiotics. The cells were incubated at 37° C in the presence of 5% CO₂. Media for both cell lines was changed every 2-3 days until confluence or the maximum cell concentration was reached. Cells were subdivided either by trypsin treatment (LLC-PK₁) to free cells from plates or after dilution in new media (CCRF-CEM).

To explore the functional implications of these findings, we compared nuclear translocation of the pro-inflammatory transcription factor NF-kB by immunofluorescent staining (Anti NF-kB p65 antibody, ab16502, Abcam, 1:200, Cambridge, MA, USA) between Lean- and MetS-MSCs. Nuclear DNA was stained with 4’,6’-diamino-2-phenylindole (DAPI, Thermo-Fisher Scientific). Double positive (NF-kB/DAPI) areas were quantified using a computer-aided image analysis program (ZEN® 2012 blue edition; Carl Zeiss SMT, Oberkochen, Germany), and the results from all fields were averaged. Furthermore, because EVs are non-nucleated, we compared the ability of Lean- and MetS-EVs to induce NF-kB translocation in pig proximal kidney tubular epithelial cells (LLC-PK1, ATCC, Manassas). Cells were cultured in Medium-199 (Gibco BRL, USA) containing 3% FBS [19] alone or co-cultured with Lean- or MetS-EVs (5μg of EV protein) for 24 hours. NF-kB/DAPI areas were quantified using ZEN®, and results from all fields were averaged. To further delineate mechanism, we evaluated expression of the TNF-α receptors (ab1939, Abcam, 1:100, Cambridge, MA), TNF-R2 (MA5-32618, Life Technologies, 1:100), the CCL20 receptor CCR6 (abPA5-29015, 1:100, Thermo-Fisher), and VEGFR1 (absc-504,1:100, Santa Cruz, USA), by immunofluorescent staining with specific antibodies.

Human and pig male proximal tubular cell lines (HK2, ATCC, CRL-2190; LLC-PK1, ATCC, CL-101) were obtained from the Cell Culture Facility (CCF) at Duke University. Wild-type and Nrf2 knockout male mouse embryonic fibroblasts (MEFs) were a generous gift from Drs. Wakabayashi, Yagishita, and Kensler (Wakabayashi et al., 2010 (link)). Cells were seeded on 96-well tissue culture assay plates at 2,500 cells/well with 3 biological replicates per condition, and they were cultured with standard methods in a humidified incubator at 37°C with 5% CO₂. HK2 and LLC-PK1 cells were cultured in DMEM high glucose (Corning) supplemented with 10% fetal bovine serum (Corning, 35–010-CV) and 1% penicillin/streptomycin (Sigma). MEFs were cultured in IMDM (Thermo, 76,050) supplemented with 10% fetal bovine serum and 1% primocin (Invivogen, ANT-PM-1) (Wakabayashietal.,2010 (link)). Cells were tested to be mycoplasma free by Duke CCF. The cell lines have not been authenticated in our lab.