HK2 cells were cultured in 24-well plates until they reached confluence. Cells were pre-treated with TAPI1 (1 µM for 30 min pre-trement, Calbiochem) and TAPI2 (10 µM for 1hr pretreatment, Cayman) and then treated with 10 µg/ml of LPS for 8 h. At the end of the experimental periods, cells were preloaded with 10 µM 2′, 7′-dichlorofluorescein diacetate (DCF-DA; Molecular Probes) for 30 min at 37℃. Fluorescence intensity was analyzed by a fluorescence reader (Fluoroscan Ascent FL; Lab systems, Helsinki, Finland) using 485 nm excitation and 538 nm emission filter. HK2 cells were cultured on a 6-well plate for DCF-DA staining. Cells were pre-treated with TAPI1 (1 µM for 30 min) and TAPI2 (10 µM for 1 h) and then treated with 10 µg/ml of LPS for 8 h. Cells were washed twice with hanks balanced salt solution (HBSS) and incubated with HBSS (without phenol red) containing DCF-DA for 30 min at 37℃ in dark. The images were obtained with a fluorescence microscope (Nikon, Tokyo, Japan).
Tapi 2
Manufactured by Cayman Chemical
Sourced in United States, United Kingdom
TAPI-2 is a chemical compound used as a laboratory reagent. It serves as a substrate for enzyme assays and other biochemical applications. The product details and technical specifications are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Fibulin 3, by OriGene (2 mentions)
Tnfα inhibitor, by Bio-Techne (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Timp3, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Bend 3, by RIKEN BioResource Center (1 mentions)
Ionomycin, by Cayman Chemical (1 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
Tcs sp8, by Leica (1 mentions)
Ab146459, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Fujifilm (1 mentions)
Sulfo nhs lc lc biotin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Hk 2 cell, by American Type Culture Collection (1 mentions)
Control sirna, by Thermo Fisher Scientific (1 mentions)
Adam10 sirna, by Thermo Fisher Scientific (1 mentions)
9 protocols using tapi 2
1
LPS-Induced Oxidative Stress Assay
2
Fibulin-3 Modulates Notch Signaling
3
Quantification of TACE activity in cells
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Transfected cells were lysed and processed for semiquantitative RT-PCR or Western blotting using standard protocols16 (link) (antibodies and primers are listed in Suppl. Tables S-I and S-II ). Cultures were treated overnight with the gamma-secretase inhibitor DAPT (25 μM, Tocris Bioscience, Bristol UK) or the TACE inhibitor TAPI-2 (1 μM, Cayman Chemical, Ann Arbor MI). Cells were also treated with TNFα (10 ng/ml, Peprotech, Rocky Hill NJ), fibulin-3 (300 ng/ml, Origene, Rockville MD), or the TNFα inhibitor C87 (10 μM, Tocris) for 1 to 24 h before collection. For reporter assays a plasmid carrying Renilla luciferase was co-transfected as loading control.
To measure TACE activity, cells were lysed in 50 mM Tricine buffer (pH 7.5) containing 100 mM NaCl, 10 mM CaCl2, 1 mM ZnCl2 and 0.1% Triton X-100. Clarified lysates (50 μg total protein) were incubated with a fluorogenic ADAM10/17 substrate peptide (TACE substrate III) as described18 (link).
To measure TACE activity, cells were lysed in 50 mM Tricine buffer (pH 7.5) containing 100 mM NaCl, 10 mM CaCl2, 1 mM ZnCl2 and 0.1% Triton X-100. Clarified lysates (50 μg total protein) were incubated with a fluorogenic ADAM10/17 substrate peptide (TACE substrate III) as described18 (link).
4
Endothelial Cell Culture and Blue Light Exposure
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bEnd.3 were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and maintained in medium formulation suggested by the American Type Culture Collection (ATCC, Manassas, VA). Primary human retinal microvascular endothelial cells (HREC) and endothelial growth medium were obtained from CellBiologics (Chicago, IL, USA). HREC were grown on gelatin-coated dishes. In the routine protocol, cell culture medium was refreshed every 2 days, and cells were passaged at 80–90% confluence. The main findings of this study were authenticated by both bEnd.3 and HREC. The knockdown of ADAM17 and GNAZ was achieved by transient transfection of the pLKO.1 vector with shRNA target sequences (RNA Technology Platform and Gene Manipulation Core, Academia Sinica, Taiwan). Stable clones were produced using puromycin selection. The TAPI-2 (ADAM17 inhibitor) was acquired from Cayman Chemical Company (Ann Arbor, MI, USA), and the stock was prepared in DMSO. The design of the blue light exposure chamber for in vitro studies is shown in Additional file 1 : Fig. S1A. Briefly, blue LEDs were oriented at the bottom of the chamber, and the illuminances (80, 160, and 240 lx) were controlled by setting the distance to the LED-emitting plate. The illuminances used in this study are common among modern 3C displayers (Additional file 1 : Fig. S1B).
5
CD80-GFP CHO Cells for T Cell Assay
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CD80-GFP CHO cells were generated by transduction with lentiviral vectors (CSII-EF-MCS) carrying CMV promotor-driven eGFP-fused mouse CD8029 . The resulting transfectants were sorted (MoFlo XDP, Beckman Coulter) for uniform GFP expression. Naïve CD4+ T cells isolated from the spleen of WT and Rap1KO mice were mixed at a 1:1 ratio with CD80-GFP donor CHO cells for 24 h in the presence of soluble anti-CD3 (5 µg/ml) or PMA (12.5 ng/ml)/ionomycin (100 ng/ml) and TAPI2 (100 µM, Cayman Chemical); BafA (25 nM, AdipoGen) was used for up to 6 h before culture termination. The mixed cells were stained with anti-CD4 and anti-CTLA-4 and analyzed by flow cytometry. The mixed cells were immobilized on poly-L-lysine-coated slides, and confocal images (TCS SP8, Leica) were obtained using a 63x objective lens.
6
Quantification of TACE activity in cells
7
HeLa Cell Surface Protein Labeling
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The human cervical carcinoma cell line HeLa was maintained in Dulbecco's modified Eagle's medium (GIBCO, CA, USA) supplemented with 10% fetal bovine serum (FBS; Wako Pure Chemical Industries, Japan), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, MO, USA) in a humidified atmosphere with 5% CO2 at 37 °C. The medium was replaced twice a week. Cell surface protein-specific labeling was performed using sulfo-cyanine3-NHS ester (sulfo-Cy3-NHS; ab146458, Abcam, UK), sulfo-cyanine5-NHS ester (sulfo-Cy5-NHS; ab146459, Abcam), Cy3-NHS ester (Cy3-NHS; IC3-OSu, Dojindo, Japan), and sulfo-NHS-LC-LC-biotin (21338; Thermo Fisher Scientific, MA, USA). Briefly, living cells were incubated with 50 μM sulfo-Cy3-NHS, sulfo-Cy5-NHS, Cy3-NHS, or sulfo-NHS-LC-LC-biotin at 4 °C for 30 min, washed with PBS, and then incubated in FBS-free medium supplemented with 15 μM ZnCl2. To inhibit ADAM17 activity, TAPI-2 (Cayman Chemical, MI, USA) was added to the medium at a concentration of 100 μM.
8
Examining HK-2 Cell Response to LPS
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Human renal proximal tubular epithelial cells (HK-2 cells, American Type Culture Collection, Manassas, VA) were cultured and passaged every 3–4 days. The detailed condition and media used for cell culture was previously described [11 (link)]. The cells were treated with or without LPS (10 µg/ml) for 8 h. The control cells were treated with a buffer solution alone. TAPI1 (1 µM for 30 min pre-trement, calbiochem, San Diego, CA, USA) or TAPI2 (10 µM for 1 h pre-treatment, Cayman, Ann Arbor, MI, USA) were used as a TACE inhibitor.
9
Recombinant HMGB1 Signaling Pathway
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Recombinant human HMGB1 (1690-HMB, endotoxin less than 0.10 EU/μg) was purchased from R&D Systems (Abingdon, UK). SB203580 and dimethyl sulfoxide (DMSO) were from EMD Chemicals (Darmstadt, Germany). TAPI-2 was from Cayman Chemical (Ann Arbor, MI, USA). Antibodies to human TLR4 (76B357.1: a mouse monoclonal antibody against amino acids 100 -200 of human TLR4), RAGE (sc-80652, a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), p38 mitogen-activated protein kinase (p38 MAPK), ADAM10 and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to human TLR4 (ab13556; a rabbit polyclonal antibody against amino acids 420-435 of human TLR4) and ADAM17 were from Abcam. An antibody to human phospho-p38 MAPK (Thr180/Tyr182) was from Cell Signaling Technology (Denvers, MA, USA).
ADAM17-siRNA, ADAM10-siRNA, RAGE-siRNA, TLR4-siRNA and control-siRNA (Ambion ® ) were from Life Technologies (Paisley, UK). The fluorogenic substrate, 5FAM-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Lys(5-TAMRA)-NH 2 , was from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). 4',6-Diamidino-2phenylindole dihydrochloride (DAPI) and Ponceau S solution were from Sigma-Aldrich (St. Louis, MO, USA).
ADAM17-siRNA, ADAM10-siRNA, RAGE-siRNA, TLR4-siRNA and control-siRNA (Ambion ® ) were from Life Technologies (Paisley, UK). The fluorogenic substrate, 5FAM-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Lys(5-TAMRA)-NH 2 , was from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). 4',6-Diamidino-2phenylindole dihydrochloride (DAPI) and Ponceau S solution were from Sigma-Aldrich (St. Louis, MO, USA).
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