The 661W cell line was generously provided by Dr. Muayyad Al-Ubaidi (Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA). This cell line is not listed as a commonly misidentified cell line by the International Cell Line Authentication Committee. Experiments utilising 661W photoreceptor cells [33 (link)] were seeded at a density of 2.3 × 104 cells/cm2 and cultured for 24 h prior to treatment. Fresh 661W medium with additional supplements (as previously described [34 (link)]) was then mixed with Poly(dA:dT) (InvivoGen, Toulouse, France) (5 µg/mL: final concentration) and added to 661W cells for up to 8 h. In the WD treatment group, 661W cells were pre-treated with 50 µM of WD for 3 h followed with Poly(dA:dT) treatment for 8 h. Ac-YVAD-cmk (100 µM) (Sigma-Aldrich, Gillingham, UK) was also pre-treated separately in 661W cells for 3 h prior to Poly(dA:dT) treatment and then for 8 h in co-treatment.
Poly da
Manufactured by Merck Group
Sourced in United Kingdom, Japan, United States
Poly(dA) is a nucleic acid polymer consisting of multiple deoxyadenylate (dA) nucleotides. Its core function is to serve as a molecular tool in various biochemical applications, such as gene expression studies and DNA/RNA synthesis.
Automatically generated - may contain errors
4 protocols using poly da
1
661W Photoreceptor Cell Activation Assay
2
Spin-labeled Tau fibrillization with RNA
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Fibrils
were formed by
incubating 35 μM spin-labeled Tau and 70 μg/mL polyA RNA
(Sigma P9403) for 3 days with stirring in 100 mM NaCl, 10 mM HEPES
buffer at pH 7.4. The RNA was polydisperse (ranging in size from ∼0.2
to 2 kb), as judged by agarose gel electrophoresis. Fibrils were also
prepared with 30–65 μM spin-labeled Tau with polyU (Sigma
P9528), double-stranded polyAU (Sigma P1537), tRNA (Sigma R8759),
or RNA extract from baker’s yeast (Sigma R650) at concentration
ranges of 140–200 μg/mL or with polydA (Sigma P0887)
at 36 μg/mL. Additionally, assembly was performed with a 4-fold
molar excess of polyglutamate (Sigma P1943) or with double-stranded
DNA, circular (pET28) or linear (Sigma D4522), at 100–200 μg/mL.
Notably, in both cases, double-stranded DNA failed to properly induce
fibrillization. Fibrils were pelleted at >100 000g for 30 min. Pellets were washed with buffer, centrifuged
further
for 10 min, and transferred to 0.60 mm i.d. × 0.84 mm o.d. borosilicate
capillaries (VitroCom CV6084-100). Samples were measured by a Bruker
EMX spectrometer fitted with an ER 4119HS resonator using a 12 mW
incident microwave power and 150 G scan width. Spectra were normalized
according to the total number of spins using double integration. A
minor background that resulted from Tau monomers (<2.0 mol %) was
subtracted from all spectra.
were formed by
incubating 35 μM spin-labeled Tau and 70 μg/mL polyA RNA
(Sigma P9403) for 3 days with stirring in 100 mM NaCl, 10 mM HEPES
buffer at pH 7.4. The RNA was polydisperse (ranging in size from ∼0.2
to 2 kb), as judged by agarose gel electrophoresis. Fibrils were also
prepared with 30–65 μM spin-labeled Tau with polyU (Sigma
P9528), double-stranded polyAU (Sigma P1537), tRNA (Sigma R8759),
or RNA extract from baker’s yeast (Sigma R650) at concentration
ranges of 140–200 μg/mL or with polydA (Sigma P0887)
at 36 μg/mL. Additionally, assembly was performed with a 4-fold
molar excess of polyglutamate (Sigma P1943) or with double-stranded
DNA, circular (pET28) or linear (Sigma D4522), at 100–200 μg/mL.
Notably, in both cases, double-stranded DNA failed to properly induce
fibrillization. Fibrils were pelleted at >100 000g for 30 min. Pellets were washed with buffer, centrifuged
further
for 10 min, and transferred to 0.60 mm i.d. × 0.84 mm o.d. borosilicate
capillaries (VitroCom CV6084-100). Samples were measured by a Bruker
EMX spectrometer fitted with an ER 4119HS resonator using a 12 mW
incident microwave power and 150 G scan width. Spectra were normalized
according to the total number of spins using double integration. A
minor background that resulted from Tau monomers (<2.0 mol %) was
subtracted from all spectra.
3
Radiolabeled DNA Synthesis Protocol
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A chemically synthesized DNA template, poly(dA), was purchased from Sigma-Aldrich Inc. and a customized oligo(dT)18 DNA primer was produced by Sigma-Aldrich Japan K.K. (Hokkaido, Japan). Radioactive nucleotide [3H]-labeled 2′-deoxythymidine-5′-triphosphate (dTTP; 43 Ci/mmol) was obtained from Moravek Biochemicals Inc. (Brea, CA, USA). All other reagents were of analytical grade from Nacalai Tesque Inc. (Kyoto, Japan).
4
Medicinal Plant Extracts for Keratinocyte Assays
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Eight extracts of medicinal plants, arnica (trade name: ARNICA EXTRACT BG), artemisia capillaris (trade name: ARTEMISIA CAPILLARIS EXTRACT BG), green tea (trade name: GREEN TEA EXTRACT), houttuynia (trade name: HOUTTUYNIA EXTRACT), marigold (trade name: MARIGOLD EXTRACT), phellodendron (trade name: PHELLODENDRON EXTRACT BG-J), rose myrtle (trade name: ROSE MYRTLE EXTRACT BG80), and turmeric (trade name: TURMERIC EXTRACT BG) were obtained from Maruzen Pharmaceuticals Co., Ltd. (Hiroshima, Japan). The solid contents of these extracts were used for experiments. The following materials were purchased from KURABO Industries Ltd. (Osaka, Japan): NHEK and serum-free keratinocyte growth medium (KGM, trade names: EpiLife-KG2 and HuMedia-KG2) containing growth additives (bovine pituitary extract, human epidermal growth factor) and hydrocortisone, insulin, and gentamycin/amphotericin B. A chemically synthesized DNA template, poly(dA), was from Sigma-Aldrich Inc. (St Louis, MO, USA), and customized oligo(dT)18 DNA primers were from Sigma-Aldrich Japan K.K. (Hokkaido, Japan). The radioactive nucleotide [3H]-labeled 2′-deoxythymidine-5′-triphosphate (dTTP; 43 Ci/mmol) was obtained from Moravek Biochemicals Inc. (Brea, CA, USA). All other reagents were analytical grade and were purchased from Nacalai Tesque Inc. (Kyoto, Japan).
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