ChIP was performed using MAGnify ChIP kit with manufacturers' instructions. Briefly, cells sorted from intestines were crosslinked with 1% formaldehyde at 37 °C for 10 min, and then quenched with 0.125 M lysine, followed by swelling in lysis buffer (50 mM Hepes, pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% NaDeoxycholate, and Protease Inhibitor Cocktail Set III) for 30 min on ice. Chromatin was sheared to a mean length of 400 bp by sonication. After being de-cross-linked by RNase, proteinase K, and heat, input genomic DNA was precipitated with ethanol and quantified in a GeneQuant 100 spectrophotometer (GE Healthcare). Chromatin was precleared with protein A/G-agarose, followed by incubation with indicated antibodies at 4 °C overnight and further incubation with protein A/G-agarose for 2 h. Beads were washed with washing buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Nadeoxycholate, 1 mM EDTA) three times and eluted with elution buffer (50 mM Tris, pH 8.0, 1% SDS, and 10 mM EDTA). Eluates were de-cross-linked by RNase, proteinase K, and heat, and DNA was extracted with phenolchloroform, followed by ethanol precipitation. For each ChIP experiment, 2 × 104 cells were used. 5% of nuclear extracts served as inputs. Immunoprecipitated DNAs were further analysed by real-time PCR. 2 × 104 NKp46+ ILC3 cells were pooled from 4–10 WT or WASH knockout mice for each group.
Genequant100 spectrophotometer
Manufactured by GE Healthcare
Sourced in United Kingdom
The GeneQuant100 is a compact, high-performance spectrophotometer designed for nucleic acid and protein analysis. It features a wide wavelength range, high-resolution optics, and intuitive software to accurately measure the concentration and purity of DNA, RNA, and proteins. The GeneQuant100 provides reliable and reproducible results for a variety of laboratory applications.
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Lab products found in correlation
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rna pre pure cell bacteria kit, by Tiangen Biotech (1 mentions)
Sybr green master mix, by Tiangen Biotech (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Lps from e coli o111 b4, by Merck Group (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Multi beads shocker, by Yasui Kikai (1 mentions)
8 protocols using genequant100 spectrophotometer
1
ChIP Assay for Intestinal ILC3 Cells
2
Chromatin Immunoprecipitation Protocol
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Cells were fixed in 1% formaldehyde for 20 min and then quenched with 0.125 M lysine, followed by swelling in lysis buffer (50 mM Hepes, pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% NaDeoxycholate, and protease inhibitors) for 30 min on ice. Chromatin was sheared to an average length of 400 bp by sonication. After being de-cross-linked by RNase, proteinase K, and heat, input genomic DNA was precipitated with ethanol and quantified in a GeneQuant 100 spectrophotometer (GE Healthcare). Chromatin was precleared with protein A/G-agarose (Santa Cruz Biotechnology, Inc.), followed by incubation with the indicated antibodies at 4°C overnight and further incubation with protein A/G-agarose for 2 h. Beads were washed with washing buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% NaDeoxycholate, 1 mM EDTA) three times and eluted with elution buffer (50 mM Tris, pH 8.0, 1% SDS, 10 mM EDTA). Eluates were de-cross-linked by RNase, proteinase K, and heat, and DNA was extracted with phenolchloroform, followed by ethanol precipitation. For each ChIP experiment, 2 × 104 cells were used. 5% of nuclear extracts served as inputs. Immunoprecipitated DNAs were further analyzed by real-time PCR. Signals were normalized to input DNA.
3
M. sprengeri Petal RNA Extraction
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The TRIzol® reagent (Invitrogen) was used to extract total RNA from the petals of red and white M. sprengeri according to the manufacturer’s instructions (Invitrogen, USA). The purity of all RNA samples was assessed at an absorbance ratio of OD260/280 and the RNA quality was tested using a 1% ethidium bromide-stained (EtBr-stained) agarose gel. A GeneQuant100 spectrophotometer (GE Healthcare, UK) assessed the RNA concentration before processing.
4
Inhibition of eNOS Oxygenase Activity
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The expression and purification of eNOS from Sf21 cells was carried out essentially same as previously reported [34 (link)]. The inhibition of eNOS oxygenase activity by unmodified or modified CBDs was tested at 37°C for 4 min by measuring L -[3H]citrulline formation in a mixture containing 25 mM Tris, pH 7.5, 100 mM NaCl, 0.5 μM CaM, 0.2 mM EDTA, 0.3 mM CaCl2, 100 μM β-NADPH, 10 μM H4B, 20 μM L -arginine, 1 μCi of L -[3H]arginine, 100 nM eNOS, and in the presence or absence of 10 μM of each peptide. Cytochrome c reduction was determined at room temperature (25°C) in a reaction mixture containing 25 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 50 μM cytochrome c, 0.5 μM CaM, 100 μM CaCl2 and 100 μM β-NADPH in the presence or absence of 10 μM of each peptide. The reaction was initiated by addition of 50 nM eNOS and monitored at 550 nm in a GE geneQuant100 spectrophotometer. Activity was determined using a ΔE550=21 mM−1cm−1. An assay in the absence of CaM was carried out to serve as baseline reference.
5
Induction of Fatty Acid Metabolism in M. pachydermatis
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The mDixon liquid medium was inoculated with one loop of M. pachydermatis and pre-cultured for 5–7 days, then transferred to 100 mL of mDixon medium for a starting OD600 of 0.05. The cells were incubated in a shaker at 120 rpm at 32 °C until OD600 reached 1–2 and then collected by washing with the mYNB (modified Yeast Nitrogen Base) medium (0.67% yeast nitrogen base (Difco), 2% glycerol, 1% Tween 40, and 25 mM MOPS buffer (pH 6)). To induce gene expression, 10 mL of cell suspension (starting OD600 = 1) in mYNB containing 5 mM of each fatty acid (C12:0, C14:0, C16:0, and C18:1) or in mDixon containing 3 μM triacsin C were incubated in L-tubes at 32 °C for 6 h. Before the cells were collected, 0.05% Triton X-100 was added as a surfactant and mixed vigorously to facilitate pellet collection. The supernatants containing each fatty acid were carefully removed, and the pellets for RNA extraction were washed with DEPC water. Total RNA was extracted from each sample using the hot phenol method [32 ]. The concentration of each isolated RNA was determined using a GeneQuant100 spectrophotometer (GE Healthcare), and RNA quality was confirmed by agarose gel electrophoresis.
6
Quantification of RB1 Gene Expression
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The total RNA from PC12 was extracted using the RNA pre-pure Cell/Bacteria Kit (Tiangen Biotech, China). The concentrations of the total RNA were measured using a Gene Quant 100 Spectrophotometer (General Electric Company, Germany). Five micrograms of total RNA were used to generate complementary DNA using an anchored oligo (dT)18 primer and EasyScript Reverse Transcriptase enzyme (Transgene, China). The cDNA was subsequently used as a template for real-time PCR using SYBR Green Master Mix (Tiangen Biotech, China) according to the manufacturer’s instructions. The primer sequences of RB1 were as follows (NCBI Reference Sequence: NM_009029.2): forward: 5′-TGCTGAAGGCGGCAATCCCC-3′ ; reverse: 5′- CGAGTCAGGTGTCCCGAGGGT-3′ .
7
Transcriptome Analysis of Old Plant Roots
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For each year, the RNA was extracted from a mixture of several old plants. Total RNA was extracted from roots using the RNeasy Plant Mini Kit (Qiagen), according to the manufacturer's instructions. The RNA quality was tested using a 1% ethidium bromide-stained (EtBr-stained) agarose gel, and the concentration was assessed using a GeneQuant100 spectrophotometer (GE Healthcare, UK) before processing. The RNA samples were treated with DNase I (TURBO DNase; Ambion, USA) at a concentration of 1.5 units/l g of total RNA prior to cDNA synthesis. The transcriptome library for sequencing was generated using the Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, San Diego, USA) following the manufacturer's recommendations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation was complete, the library preparations were sequenced on an Illumina Hiseq 2000 platform and 100 bp paired-end reads were generated.
8
LPS-induced gene expression in cells
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BIE and MAC-T cells were seeded in 6-well plates at densities ranging from 4–7 ×
105 cells and incubated for 15 ± 1 hr before experiments. After incubation,
the cells were rinsed with DMEM without FBS and then treated with 0–100
µg/ml LPS from E. coli O111:B4
(L2630, Sigma) in DMEM without FBS at 37°C for 2 hr. The cells on the plates were
collected, washed with PBS, and total RNA was immediately extracted from the cells using
the RNeasy Mini kit according to the manufacturer’s instructions. Thirty milligrams each
of mucosal epithelial tissue samples (small intestine, mammary gland, lung, and uterus)
and liver were homogenized with 600 µl of RLT buffer containing
β-mercaptoethanol from the RNeasy Mini Kit by using Multi-Beads Shocker (Yasui Kikai,
Osaka, Japan), and the total RNA was extracted as described above.
The RNA extracted from cell lines and tissue samples was quantified using a GeneQuant 100
spectrophotometer (GE Healthcare) and stored at −80°C until use. Contaminating DNA was
removed with DNase I (18068-015, Invitrogen), and cDNA was synthesized using PrimeScript
RT Master Mix (RR036A, Takara, Kusatsu, Japan) according to the manufacturer’s
instructions.
105 cells and incubated for 15 ± 1 hr before experiments. After incubation,
the cells were rinsed with DMEM without FBS and then treated with 0–100
µg/ml LPS from E. coli O111:B4
(L2630, Sigma) in DMEM without FBS at 37°C for 2 hr. The cells on the plates were
collected, washed with PBS, and total RNA was immediately extracted from the cells using
the RNeasy Mini kit according to the manufacturer’s instructions. Thirty milligrams each
of mucosal epithelial tissue samples (small intestine, mammary gland, lung, and uterus)
and liver were homogenized with 600 µl of RLT buffer containing
β-mercaptoethanol from the RNeasy Mini Kit by using Multi-Beads Shocker (Yasui Kikai,
Osaka, Japan), and the total RNA was extracted as described above.
The RNA extracted from cell lines and tissue samples was quantified using a GeneQuant 100
spectrophotometer (GE Healthcare) and stored at −80°C until use. Contaminating DNA was
removed with DNase I (18068-015, Invitrogen), and cDNA was synthesized using PrimeScript
RT Master Mix (RR036A, Takara, Kusatsu, Japan) according to the manufacturer’s
instructions.
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