Trypsin ethylenediaminetetraacetic acid edta

RAW 264.7 and THP-1
macrophage cells were obtained from the American Type Culture Collection
(ATCC, U.S.A.). Jurkat JE6-1 TLR 4, TLR 6, TLR 2/1, TLR 2/6 and TLR
2/1/6 were a kind gift from Peter Steinberg’s Lab (Medical
University of Vienna). Hank’s balanced salt solution (HBSS),
Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park
Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), phorbol
12-myristate 13-acetate (PMA) and ionomycin (iono) was purchased from
Sigma-Aldrich. Trypsin-ethylenediaminetetraacetic acid (EDTA) was
purchased from Invitrogen, U.S.A.

The TECs were primary isolated from the kidneys of both WT and P^KO C57BL/6 male mice aged 8–12 weeks using a well-established method that led to the functional characterization of TECs in the group (30 (link)). Briefly, small cortical pieces from the kidney were pulverized and passed through a serial set of sieves with the size from 250, 125, 75, and 45 μm. The primary isolates were maintained in DMEM/F-12 medium containing 10% heat-inactivated fetal bovine serum (FBS, Sigma) and other basic additives as the same for TCMK-1 cells, but with additional recombinant human epithelial growth factor (0.1 µg/ml), insulin (5 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml), triiodothyronine (4 pg/ml), and hydrocortisone (36 ng/ml). At confluence, TECs (passage 0) were split using trypsin/ethylenediaminetetraacetic acid (EDTA) (Invitrogen), and cells at passages 2–3 were used for experiments. The characterization of isolated TECs was previously validated by the positive staining of cytokeratin and γ-glutamyl transpeptidase and the negative staining of factor VIII and α-smooth muscle actin that are the markers of endothelial cells and mesangial cells, respectively (30 (link)). In addition, the morphological characteristics of newly isolated and cultured TECs that formed domes are illustrated in Supplementary Figure 1 (31 (link)).

For the human lung adenocarcinoma cell line NCI-H1299, obtained from the American Type culture collection (ATCC), VA was first stably transfected with green fluorescent protein (GFP) protein (H1299-GFP). This modified cell line was then used instead of actual CTCs as a model to evaluate the reliability of the developed device. The cells were cultured according to the protocols from ATCC in DMEM/HIGH GLUCOSE medium supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37 °C with 5% CO_2. The medium was changed every 1–2 days of culturing. Cells were trypsinized by incubation in 0.05% Trypsin-ethylene diamine tetra acetic acid (EDTA) (Invitrogen, CA) at 37 °C for 2 min to suspended them and subsequently dilute them to the desired concentration.

Human melanoma cell line A375, human lung carcinoma cell lineA549, human cervical squamous carcinoma cell line HeLa, human hepatoma cell line Hep G2, human colon carcinoma cell line HT-29, human breast carcinoma cell line MCF-7, and human gastric adenocarcinoma cell line MKN-28 were purchased from the American Type Culture Collection (ATCC) and Dr. Murakami’s Research Laboratory, Kyushu University, Japan. Dulbecco’s modified Eagle’s medium (DMEM) and trypsin- ethylenediaminetetraacetic acid (EDTA) were obtained from Invitrogen, California, USA. Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). Aquaresin Turmeric was purchased from KALSCE®, Michigan, USA. Sodium silicate solution was obtained from Wako Pure Chemical, Osaka, Japan. Chitosan was obtained from LYTONE Enterprise. Inc., Taipei, Taiwan. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich, Missouri, USA. Lactate dehydrogenase (LDH) leakage kit was acquired from Promega, Wisconsin, USA. PE-conjugated anti-human DR5 was purchased from ebioscience, San Diego, CA, USA. Ferrous chloride and ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4’,4’’-disulfonic acid sodium salt) were purchased from Sigma, St. Louis, MO, USA.