Defined trypsin inhibitor

Primary PTECs from WT and KO mice were isolated under sterile conditions using previously described methods with a few modifications^{64 (link),65 (link)}. Renal cortices were dissected visually in ice-cold HBSS and sliced into small pieces. The fragments were digested in PBS buffer containing 1 mg/ml type I collagenase (Worthington, Lakewood, NJ) and 125 μg/ml defined trypsin inhibitor (Gibco) at 37 °C for 30 min. The supernatant was sieved through two nylon sieves (pore size 200 um and 70 um). Tubular fragments caught by the 70-μm sieve were flushed in the reverse direction with PBS and centrifuged for 5 min at 200 × g. Thereafter the pellet was resuspended in DMEM/F12 medium, overlayed on an OptiPrep^TM density gradient solution (Sigma-Aldrich) and centrifuged for 20 min at room temperature 800 × g^{66 (link)}. Highly purified proximal tubules in the fraction between 1.05 and 1.076 g/ml were collected^{67 (link)}, washed twice in ice cold PBS and maintained in DMEM/F12 medium supplemented with 5% FBS, 1× MEM nonessential amino acids, 1 × insulin-transferin-selenium, 100 IU/ml penicillin and 100 ug/ml streptomycin. The plate was incubated in a humidified incubator under 5% CO₂ at 37 °C. The medium was changed every other day until 90% of cell cultures were organized as a confluent monolayer.

The fresh GBM tissue obtained from surgical tumor resection was processed according to previously published protocol [16 ]. Shortly, the tissue pieces were minced carefully and washed with DMEM/F12 medium supplemented with 100 IU/ml penicillin and 50 mg/ml streptomycin. After washing the medium was replaced with 5 ml Trypsin EDTA (Gibco) and incubated at +37 °C for 30 min. The detachment of cells was ensured by vigorous pipetting up and down every 10 min. The trypsin was inhibited by adding 10 ml of Defined trypsin inhibitor (Gibco). The non-dissociated tissue pieces were allowed to sediment and the medium containing single cells was moved into a new tube. The cells were collected by centrifuging 1000 rpm 5 min. Cells were resuspended into DMEM/F12 supplemented with 100 IU/ml penicillin, 50 mg/ml streptomycin, EGF 20 ng/ml (R&D systems), FGF 20 ng/ml (R&D systems) and vitamin A depleted B27 supplement (Gibco) in a density of 50 000 cells/ml and plated into cell culture flasks. The flask were incubated in cell culture incubator for 7 days for neurospheres to form. The culture medium was changed every second day. The neurospheres were trypsinized and plated into new cell culture flasks every second week.

Cells were washed once with PBS after treatment or infection following which they were detached using trypsin (80–120 μL) (Gibco) and collected by adding defined trypsin inhibitor (Gibco). Cells were resuspended in DMEM without phenol red (Gibco) supplemented with 2 mM L-glutamine (Gibco) (staining media). Cells were stained using either 5 μM Fluozin-3-AM (Molecular Probes) or 2.5 μM ZinPyr-1 (ZP-1) (Santa Cruz) in the staining media. For ZP-1 staining, medium containing 1 mM EDTA was added to chelate any extracellular zinc during staining. Cells were incubated for 30 min at 37°C in CO₂ incubator and mixed every 10 min. For assessing cell viability, fixable viability stain eFluor780 (Becton Dickinson Biosciences) was added to the cells at 1:500 dilution prepared in the staining medium and incubated for further 10 min at 37°C in the CO₂ incubator. Cells were washed using FACS buffer (PBS containing 0.25% FBS) and acquired in FACS Canto II (Becton Dickinson). The amount of labile zinc present in live cells was presented as the mean fluorescence intensity of Fluozin-3-AM and ZP-1. Images were quantitated using cellSens Software (Olympus).

MRL/Lpr mice splenic T cells were purified with Dynabeads FlowComp Mouse Pan T (CD90.2) Kit and dissociated from beads according to manufacturer's instructions (Thermo Fisher). After staining purified T cells with PE anti-CD138 antibody, TCRβ+CD138− and TCRβ+CD138+ cells were further separated with anti-PE magnetic microbeads (Miltenyi Biotec). Depending on the experimental objective, purified TCRβ+CD138+ and TCRβ+CD138− cells were stimulated with 1 μg/ml anti-CD3 and anti-CD28 antibodies (BD Pharmingen), 10 ng/ml PMA and 100 ng/ml Ion, 100 mg/ml collagenase I, 100 mg/ml collagenase D, 2.5 mg/ml trypsin, 10 μM leupeptin, 500 μM aminophenyl mercuric acetate (all from Sigma–Aldrich), 10 μM focal adhesion kinase inhibitor 14 (Cayman chemicals), 10 μg/ml MMP9, TrypLE, or defined trypsin inhibitor (all from Thermo Fisher) for indicated duration, and the expression levels of cell surface CD138 were quantified by fluorescence-activated cell sorting (FACS). trypsin gene expression levels were quantified by quantitative PCR, and trypsin protein was analyzed by Western blot using rabbit polyclonal trypsin antibody (Thermo Fisher).