253G1‐hiPSCs 43 (provided by Prof. Shinya Yamanaka at CiRA, Kyoto University) were transduced with the Tet‐inducible HSVtk lentiviral vector at a multiplicity of infection (MOI) of 2–10. Almost 100% transduction efficiency was observed based on examining humanized Kusabira‐Orange 1 fluorescent protein (hKO1) 44 expression under a fluorescence microscope. Single hKO1‐positive iPSCs were sorted using the FACSAria III system (BD Biosciences, San Jose, CA) and then expanded. 253G1‐hiPSCs expressing Tet‐inducible HSVtk (HSVtk‐hiPSCs) were dissociated into single cells, seeded in 96‐well plates at a density of 5 × 103 cells/200 μl per well with or without 1 μg/ml doxycycline (DOX; Wako Pure Chemical Industries, Ltd., Osaka, Japan). After 3 days of incubation, the cell viability assay was performed using the Cell Counting Kit‐8 (Dojindo Molecular Technologies, Kumamoto, Japan) as described previously 41 .
Doxycycline dox
Manufactured by Fujifilm
Sourced in Japan
Doxycycline (DOX) is a laboratory product manufactured by Fujifilm. It is a broad-spectrum tetracycline antibiotic that inhibits bacterial protein synthesis. DOX is commonly used in research applications as a selective agent in cell culture and molecular biology experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facs aria 3 system, by BD (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Tet plko puro, by Addgene (1 mentions)
Puromycin, by Merck Group (1 mentions)
Glycerol, by Fujifilm (1 mentions)
Tezacaftor, by Selleck Chemicals (1 mentions)
Elexacaftor, by Selleck Chemicals (1 mentions)
Cycloheximide (chx), by Fujifilm (1 mentions)
Lumacaftor, by Cayman Chemical (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mg132, by Cayman Chemical (1 mentions)
3 protocols using doxycycline dox
1
Inducible Suicide Gene in hiPSCs
2
LRRK2 Silencing in HL-60 Cells
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LRRK2 was silenced by infecting HL-60 cells with lentiviruses constructed by inserting Lrrk2 shRNA sequence (sh1: TRCN0000021462; sh2: TRCN0000021460, Merck) into Tet-pLKO-puro (Addgene, Watertown, MA, USA). Tet-pLKO-puro was a gift from Dr. Dmitri Wiederschain [21 (link)]. The targeting sequence of the irrelevant (Irr) shRNA has been previously reported [22 (link)]. The infected HL-60 cells were selected with 0.8 µg/ml puromycin (Merck). For gene silencing in dHL-60 cells, HL-60 cells expressing tetracycline-inducible Lrrk2 shRNA or Irr shRNA were cultured in the presence of 1.25% DMSO for 1 day, followed by 3 days in a medium supplemented with 1.25% DMSO and 0.5 µg/ml doxycycline (Dox; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
3
Screening of CFTR Modulator Compounds
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The following chemicals were used: DMSO (Sigma-Aldrich, St Louis, MO, Cat# D2650), MG-132 (Cayman Chemical, Ann Arbor, MI, Cat# 10012628), Lumacaftor (Cayman Chemical, Cat# 22196), Tezacaftor (Selleck Chemicals, Houston, TX, Cat# S7059), Elexacaftor (Selleck Chemicals, Cat# S8851), Ivacaftor (Chemscene LLC, Monmouth Junction, NJ, Cat# CS-0497), cycloheximide (CHX, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, Cat# 3720991), doxycycline (dox, FU-JIFILM Wako Pure Chemical Corporation, Cat# 049-31121), glycerol (FU-JIFILM Wako Pure Chemical Corporation, Cat# 075-00616), cyclosporin A (CLP-A, FU-JIFILM Wako Pure Chemical Corporation, Cat# 031-24931), E-4031 (Selleck Chemicals, Cat# S6627). Hit compounds from the in silico screening and the FR3 analog compounds are listed in Supplementary Table S1 .
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