A live cell, high-throughput, microplate-based ROS assay that utilized the cell permeable substrate, 2',7'-dichlorofluorescin diacetate, (DCF-DA) a reliable fluorogenic marker for ROS detection was used as previously described [10] . Upon ROS generation, the highly fluorescent dye, 2',7'-dichlorofluorescein is produced, with EX (excitation): 495 nm and EM (emission): 530 nm. In brief, DLD-1.ApoL6 cells were plated in 96-well tissue culture plates at a density of 100,000 cells/well and then loaded with DCF-DA at 20 μM final concentration for 30 min before treating with the indicated compounds at 2, 10 and 50 μM in D.0 for 8 hrs. Both end point and area-scan readings were taken using the Synergy H4 Plate Reader at 485 nm with emission at 528 nm using a 20 nm bandwidth, according to the manufacturer's instruction.
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Cells were plated at a density of 1×104 cells in 6 replicates in 96-well dish. Twelve hours later, the cells were incubated with a cocktail of 5 μM concentration of CellROX Deep Red Reagent (C10422, Invitrogen) and 2 μg/ml Hoechst 33342(Invitrogen) for 45 minutes at 37°C. Images were captured using a Zeiss fluorescent microscope and Axiovision Image 4.5 software. Quantification of the percent ROS was obtained using a high-throughput immunofluorescence plate reader (Celigo)40 (link).
Cells
Fluorescent Antibody Technique
HOE 33342
Hyperostosis, Diffuse Idiopathic Skeletal
Microscopy
TEM was performed as described previously with some modifications (Yuan et al., 2013 (link)). Briefly, small pieces of WT and miR-dKO testes were fixed in 0.1 M cacodylate buffer (pH 7.4) containing 3% paraformaldehyde and 3% glutaraldehyde plus 0.2% picric acid for 2 h in 4°C, then for 1 h at RT. Following washes with 0.1 M cacodylate buffer, the samples were post-fixed with 1% OsO4 for 1 h at RT. Dehydration was performed using 30%, 50%, 70%, 90% and 100% ethanol solutions sequentially, followed by infiltration of propylene oxide and Eponate with BDMA overnight at RT. After infiltration, samples were embedded in Eponate mixture (Electron Microscopy Sciences, Hatfield, PA, USA) and polymerized at 60°C for 24 h. Ultrathin sections (60–70 nm in thickness) were cut with a diamond knife using an ultra-microtome (Leica). The sections were collected on collodion covered electron microscope nickel grids and stained with uranyl acetate and lead citrate. The ultrastructure of the samples was observed and photographed using a transmission electron microscope (Phillips CM10) at 80 kV.
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1-naphthol-8-amino-3,6-disulfonic acid
Buffers
Cacodylate
Citrate
Collodion
Dehydration
Diamond
Electron Microscopy
Ethanol
Glutaral
Microtomy
N-benzyl-N,N-dimethylamine
Nickel
paraform
propylene oxide
Testis
Transmission Electron Microscopy
uranyl acetate
JTP-74057, a highly specific and potent MEK1/2 inhibitor [35 (link)], and GDC-0994, a highly selective ERK1/2 inhibitor [36 (link)], were used to prevent the activation of the MEK/ERK pathway. Cells were treated with 1 nmol/L JTP-74057 or GDC-0994 in a humidified incubator for 24 h at 37 °C with 5% CO2.
Doxorubicin is a chemotherapy medication used to treat cancer. Cells were treated with 5 μmol/L doxorubicin for 24 h, followed by the cell counting kit-8 (CCK-8) assay and western blotting.
Doxorubicin is a chemotherapy medication used to treat cancer. Cells were treated with 5 μmol/L doxorubicin for 24 h, followed by the cell counting kit-8 (CCK-8) assay and western blotting.
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Biological Assay
Cells
Doxorubicin
GDC-0994
JTP 74057
Malignant Neoplasms
MAP2K1 protein, human
MEK-ERK Pathway
Mitogen-Activated Protein Kinase 3
Pharmaceutical Preparations
Pharmacotherapy
The following antibodies were used for immunofluorescence (IF) and immunoblotting (IB): mouse antibodies against ACTB/β-ACTIN (Sigma-Aldrich, A5441; IB, 1:10,000), DDIT3 (Cell Signaling Technology, 2895; IB, 1:1,000), NFKBIA (Cell Signaling Technology, 4814; IB, 1:1,000), VPS37A (Santa Cruz Biotechnology, sc-376978; IB, 1:100); rabbit antibodies against ATF3 (Cell Signaling Technology, 18665; IB, 1:1,000), ATF4 (Cell Signaling Technology, 11815; IB, 1:1,000), ATG5 (Cell Signaling Technology, 12994; IB, 1:1,000), ATG7 (Cell Signaling Technology, 8558; IB, 1:1,000), BCL2L1 (Cell Signaling Technology, 2762; IB, 1:1,000), HSPA5/BiP (Cell Signaling Technology, 3177; IB, 1:1,000), CHMP2A (Proteintech, 10477-1-AP; IB, 1:1,000), cleaved CASP3 (Cell Signaling Technology, 9661; IB, 1:1,000), cleaved CASP8 (Cell Signaling Technology, 9496; IB, 1:1,000), CASP9 (Cell Signaling Technology, 9502; IB, 1:1,000), TNFRSF10B/DR5 (Cell Signaling Technology, 8074; IB, 1:1,000), GAPDH (Proteintech, 60004-1-lg; IB, 1:50,000), HRK (Novus, NBP1-76414; IB, 1:400), LMNB1 (Proteintech, 12987-1-AP; IB, 1:5,000), MAP1LC3B (Novus, NB100-2220; IB, 1:5,000) (Cell Signaling Technology, 3868; IF, 1:200), PARP (Cell Signaling Technology, 9542; IB, 1:1,000), PMAIP1 (Cell Signaling Technology, 14766; IB, 1:1,000), CASP8 (Abcam, ab108333; IB, 1:1,000), RELA (Cell Signaling Technology, 8242; IB, 1:1,000), TRIB3 (Proteintech, 13300-1-AP; IB, 1:1,000); guinea pig antibody against SQSTM1/p62 (American Research Products, 03-GP62-C; IB, 1:4,000; IF, 1:400). SMARTvector Inducible Lentiviral shRNAs and ON-TARGETplus SMART Pool siRNAs listed in Table S7 were purchased from Horizon Discovery. sgCASP8-Cas9-2A-GFP plasmid was purchased from Santa Cruz Biotechnology (sc-400147). epiCRISPR (135960) [52 (link)], LentiCRISPR v2 (52961) [53 (link)], LRG (Lenti_sgRNA_EFS_GFP, 65656) [54 (link)] and pSI-Check2-hRluc-NFkB-firefly (106979) [55 (link)] were obtained from Yongming Wang, Feng Zhang, Christopher Vakoc and Qing Deng, respectively, through Addgene. For CRISPR-Cas9-mediated gene silencing, each gRNA listed in Table S5 was subcloned into the BsmB1 site of lentiCRISPRv2 and the Sap1 site of epiCRISPR. LRG plasmids encoding three distinct VPS37A gRNAs (LRG-gVPS37As) were generated as previously described [20 (link)] and used to generate ATG7 VPS37A and CASP8 VPS37A DKO cells. pCDH1-myc-CASP8(C360A)-KN151-SV40-hygro and pCDH1-HA-CASP8(C360A)-LC151-SV40-hygro were previously described [19 (link)]. All other reagents were obtained from the following sources: 5Z-7-oxozeaenol (MedChemExpress, HY-12686); 7-amino-actinomycin D (7-AAD; BioLegend, 420404); APC Annexin-V (BioLegend, 640941); BMS-345541 (Sigma-Aldrich, B9935); bovine serum albumin (BSA; EMD Millipore, 126575); Caspase-3/7 Apoptosis Assay Reagent (Essen Biosciences, 4704); Caspase-Glo 3 Assay kit (Promega, G8090); Caspase-Glo 8 Assay kit (Promega, G8200); deoxycholate (Sigma-Aldrich, D6750); digitonin (Sigma-Aldrich, 11024-24-1); dimethyl sulfoxide (DMSO; Sigma-Aldrich, D2438); iTaq Universal SYBR Green Supermix (BIO-RAD, 1725121); Dual-Glo Luciferase Assay System (Promega, E2920); Dulbecco’s Modification of Eagle’s Medium (DMEM; CORNING, 10–013-CV); GSK2656157 (MedChem Express, HY-13820); Incucyte Caspase-3/7 Red Dye (Essen Biosciences, 4704); jetPRIME (Polyplus, 101000015); Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778075); NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); normal goat serum (Sigma-Aldrich, G9023); paraformaldehyde (Electron Microscopy Sciences, 15710); Phosphatase Inhibitor Cocktail 2/3 (Sigma-Aldrich, P5726/P0044); Protease Inhibitor Cocktail (Sigma-Aldrich, P8340); QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, 015); QuantiTect Reverse Transcription Kit (Qiagen, 205311); RNeasy Plus Mini Kit (Qiagen, 74134); Thapsigargin (Sigma-Aldrich, T9033); Triton X-100 (Sigma-Aldrich, T8532); tunicamycin (Sigma-Aldrich, T7765); YOYO-3 iodide (Thermo Fisher Scientific, Y3606).
Cells were plated in a 24 well plate (Corning, Corning, NY) and were allowed to adhere over night, at 37°C and 5% CO2. The following day, apoptosis was induced by several methods: Staurosporine- cells were incubated with 0.5 µM Staurosporine (Sigma-Aldrich) for 1.5 h and then washed with fresh media. γ-Irradiation- cells were irradiated (10Gy in GammaCell 220 Excel, MDS Nordion). Peroxide treatment- medium was aspirated and cells were washed with PBS with Ca2+ and Mg2+ (Biological Industries). Cells were incubated with H2O2 (30%, Sigma, Steinheim, Germany) diluted in PBS to a final concentration of 200 µM, at 37°C. After 1 h, cells were washed and fresh medium was added. Anti-FAS mAb- cells were treated with 1 µg/ml of anti-human FAS mAb (MBL, Naka-ku Nagoya, Japan). The number of apoptotic cells in the cultures was determined at different time points following apoptosis induction using Annexin/PI kit (MBL) according to manufacturer instructions.
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Annexins
Apoptosis
Biopharmaceuticals
Cells
Homo sapiens
Peroxide, Hydrogen
Peroxides
Staurosporine
Liver specimens from mice that were injected i.v. with the green lipophilic dye PKH67 (Sigma, St Louis, MO, USA)-labelled RL-male-1 cells (5 × 10 6 ) and either LAD-1 (200 µg) or control rat IgG (200 µg) were fixed in periodate-lysine-paraformaldehyde fixative, frozen, and sections cut (8 µm) with a cryostat as described previously [17] . The sections were blocked with phosphate buffered saline (PBS) containing 5% bovine serum albumin, and incubated for 6 h at 4°C with biotinylated anti-CD80 mAb and for 30 min at room temperature with Texas red-avidin (BD Pharmingen). A biotinylated hamster anti-KLH mAb (BD Pharmingen) was used as a negative control. The sections were examined by fluorescence microscopy (Olympus, Tokyo, Japan). Peritoneal exudate cells (PEC), obtained by rinsing the peritoneal cavity with PBS, were cultured for 3 h with PKH67labelled RL-male-1 cells at a ratio of 1 : 1 in the presence of either LAD-1 or control M17/4 or 30-H12 IgG (10 µg each). The cells were then harvested and labelled with anti-CD80 mAb, as described above. In some experiments, cells were labelled with PE-anti-CD80 mAb and analysed with a confocal microscope as described previously [18] as well as by flow cytometry.
Chromogenic immunohistochemistry was performed mainly using the automated Ventana Discovery ULTRA (Roche). The slides were steamed for 32 min in CC1 buffer (950-124, Roche) and incubated in the corresponding primary antibody at room temperature for 36 min. Anti-MTCO1 antibody was used at 1:8000 dilution (ab14705, abcam, Cambridge, MA) and Anti-COX IV antibody was used at 1:8000 dilution (3E11, 4850, Cell Signaling, Danvers, MA) as primary antibodies. The slides were developed using Discovery HQ HRP hapten-linked multimer detection kit (Roche).
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IHC was performed on 4 µm formalin-fixed, paraffin-embedded tissue sections using BRG1 (SMARCA4), AR, FOXA1, ERG and C-Myc. The IHC process was performed on the Ventana ULTRA automated slide staining system using the Omnimap and Ultraview Universal DAB detection kit. The antibody details are provided in SI Appendix, Table S1 . The following commercial kits from Roche-Ventana Medical System were used: Discovery CC1 (Cat No. 950-500), Discovery CC2 (Cat No. 950-123), OptiView Universal DAB Detection Kit (Cat No. 760-700), and OmniMap Universal DAB Detection Kit (Cat No. 760-149).
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The venom gland tissue was homogenized in a 1 ml glass homogenizer with TRIzol solution (Invitrogen, Calsbad, CA, USA) aseptically. Then, 20% chloroform was added, and the sample was centrifuged and treated with RNA-free DNAase I to separate RNA from the cellular debris and residual DNA. The separated RNA was then pelleted using isopropyl alcohol and washed with 75% ethanol. The polyadenylated mRNA (poly(A)+ mRNA) was purified with oligo (dT) magnetic beads from 20 μg of total RNA, as per manufacturer’s instructions (Illumina, San Diego, CA, USA). The quality of the purified RNA was assessed immediately using the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). The RNA integrity number (RIN) of the sample was determined to be 8.6, indicating that the RNA was in good condition for downstream transcriptomic analysis.
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Cells
Chloroform
Deoxyribonucleases
Ethanol
Gene Expression Profiling
Isopropyl Alcohol
mRNA, Polyadenylated
Oligonucleotides
RNA, immune
RNA I
Tissues
trizol
Venoms
IHC was carried out using the protocol highlighted in the Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit (ABCAM ab64264). Briefly, after rehydration, slides were subjected to antigen retrieval by boiling in citrate buffer (antigen unmasking solution, citric acid based, Vector) and allowed to slowly cool to room temperature. Slides were washed in PBS and blocked (protein, and then hydrogen peroxide block), and then incubated with primary antibody overnight (4°C). Slides were washed and then incubated secondary (anti‐rabbit/anti‐mouse biotinylated) and tertiary antibodies (streptavidin‐HRP) to label targets with HRP, incubated for 30 seconds to 1 minute with DAB substrate. Slides were counterstained with hematoxylin and coverslips were added before imaging.
Antibodies
Antigens
Buffers
Citrate
Citric Acid
Cloning Vectors
Hematoxylin
Immunoglobulins
Mice, House
Neoplasm Metastasis
Peroxide, Hydrogen
Proteins
Rabbits
Rehydration
Streptavidin
DAB (3,30-diaminobenzidine) single-plex IHC detection was carried out initially to determine the appropriate primary antibody conditions using the Ventana Discovery Ultra automated IHC/ISH research platform (Roche Diagnostics Limited, Burgess Hill, UK) before translating to the TSA immunofluorescence.
Freshly cut 4 μm tissue sections were mounted on glass slides. Sections were placed in the Ventana Discovery Ultra and dewaxed; heat induced epitope retrieval was performed using CC1 reagent for 64 min. Primary antibodies were added by manual application and the final concentrations determined are provided in Table1 . Secondary HRP complexes and DAB were completed in a fully automated manner. Slides were washed, dehydrated through a series of alcohols and coverslipped. Slides were imaged using an Aperio CS2 whole slide scanner.
Freshly cut 4 μm tissue sections were mounted on glass slides. Sections were placed in the Ventana Discovery Ultra and dewaxed; heat induced epitope retrieval was performed using CC1 reagent for 64 min. Primary antibodies were added by manual application and the final concentrations determined are provided in Table
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Antibodies
Diagnosis
Epitopes
Ethanol
Fluorescent Antibody Technique
Immunoglobulins
Tissues
The recombinant GST or His tagged proteins were expressed in E. coli strain BL21‐CodonPlus (DE3) with 1 mM IPTG for 4 h at 37°C. The cells were collected and lysed in PBS (pH 7.4) supplemented with 0.1% Triton‐100, 1 mM PMSF and 1 mg/ml lysozyme. After sonication on ice for 30 min, the samples were centrifuged, and the supernatants were purified using a His‐tag protein purification kit (Cat. no. P2226; Beyotime) and a GST protein purification kit (Cat. no. P2262; Beyotime).
Cells
Escherichia coli
Isopropyl Thiogalactoside
Muramidase
Proteins
Strains
Antibodies, Anti-Idiotypic
Immunoprecipitation
RNA-Binding Proteins
ALKBH5 RIP was carried out using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Sigma, 17-700) according to the manufacturer's protocol. 3.5 mg of anti-ALKBH5 antibody (Sigma, HPA007196) was used for each reaction, with the same amount of IgG used as a control.
Nucleofection was performed in all experiments using K562, HeLa, and
U2OS cells. For PE conditions in these cell types, 800ng prime editor-expression
plasmid, 200ng PEgRNA-expression plasmid, and 83ng nicking plasmid was
nucleofected in a final volume of 20uL in a 16-well nucleocuvette strip (Lonza).
For HDR conditions in these three cell types, 350 ng nuclease-expression
plasmid, 150 ng sgRNA-expression plasmid and 200 pmol (6.6 μg) 100-nt
ssDNA donor template (PAGE-purified; Integrated DNA Technologies) was
nucleofected in a final volume of 20 μL per sample in a 16-well
Nucleocuvette strip (Lonza). K562 cells were nucleofected using the SF Cell Line
4D-Nucleofector X Kit (Lonza) with 5 × 105 cells per sample
(program FF-120), according to the manufacturer’s protocol. U2OS cells
were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with
3—4 × 105 cells per sample (program DN-100), according
to the manufacturer’s protocol. HeLa cells were nucleofected using the SE
Cell Line 4D-Nucleofector X Kit (Lonza) with 2 × 105 cells per
sample (program CN-114), according to the manufacturer’s protocol. Cells
were harvested 72 hours after nucleofection for genomic DNA extraction.
U2OS cells. For PE conditions in these cell types, 800ng prime editor-expression
plasmid, 200ng PEgRNA-expression plasmid, and 83ng nicking plasmid was
nucleofected in a final volume of 20uL in a 16-well nucleocuvette strip (Lonza).
For HDR conditions in these three cell types, 350 ng nuclease-expression
plasmid, 150 ng sgRNA-expression plasmid and 200 pmol (6.6 μg) 100-nt
ssDNA donor template (PAGE-purified; Integrated DNA Technologies) was
nucleofected in a final volume of 20 μL per sample in a 16-well
Nucleocuvette strip (Lonza). K562 cells were nucleofected using the SF Cell Line
4D-Nucleofector X Kit (Lonza) with 5 × 105 cells per sample
(program FF-120), according to the manufacturer’s protocol. U2OS cells
were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with
3—4 × 105 cells per sample (program DN-100), according
to the manufacturer’s protocol. HeLa cells were nucleofected using the SE
Cell Line 4D-Nucleofector X Kit (Lonza) with 2 × 105 cells per
sample (program CN-114), according to the manufacturer’s protocol. Cells
were harvested 72 hours after nucleofection for genomic DNA extraction.
Cell Lines
Cells
Genome
HeLa Cells
K562 Cells
Plasmids
Tissue Donors
The test for ammonia production was carried out following the protocol of Singh et al. (2014) [31 (link)]. Briefly, each microbial isolate was grown in 5 ml of peptone water for 72 h at 30 °C on a shanking incubator (3 g). Microbial cells were removed by centrifugation at 12,298 g for 5 min and Nessler's reagent (K2HgI4; 1.4%) was added in 2:1 ratio to each culture supernatant. The development of faint to deep yellow to brownish colour indicated the production of ammonia and it was quantified by measuring the absorbance of mixture (peptone broth culture + Nessler's reagent) at 450 nm. Standard curve between different concentrations of ammonium chloride (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 mM/ml) and absorbance was used to compute the concentration of ammonia produced by the isolates.
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Amines
Buffers
Dialysis
Dimethylformamide
Ethanol
Ethyl Ether
Lipids
Molar
Polymers
RNA, Messenger
Sodium Acetate
Sulfoxide, Dimethyl
Buffers
Esters
Glucose
Lipofectamine
Liposomes
poly(beta-amino ester)
Poly A
Polymers
RNA, Messenger
Serum
Sunlight
Transfection
Bath
Biological Assay
Chondrogenesis
Chondroitin 4-Sulfate
dimethylmethylene blue
DNA, Double-Stranded
Fluorescence
Medical Devices
Molecular Probes
Papain
PicoGreen
BM-MSC and SM-MSC pellets were collected and stored at -20 °C in medium prior to biochemical assays. The dimethylmethylene blue (DMMB) spectrophotometric assay (Sigma-Aldrich, St. Louis, MO) was used to quantify proteoglycan content in pellets digested in 0.5 mg/mL papain (Sigma Aldrich St. Louis, MO). Chondroitin-4 sulfate (Sigma-Aldrich, St. Louis, MO) was used to establish a standard curve and the optical density determined at 525 nm [47 (link)]. Total DNA content was determined using 0.5 mg/mL papain digested pellets incubated for 24 h at 65 °C. Digested samples were then mixed bisbenzimide compound (Hoescht, Sigma-Aldrich, Burlington, MA) and DNA was quantified using a fluorometric assay with an excitation wavelength of 348 nm and an emission wavelength of 456 nm. Calf thymus DNA (Sigma-Aldrich, St. Louis, MO) was used to establish a standard curve. Proteoglycan concentration was normalized to the quantity of DNA in that sample.
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Biological Assay
Bisbenzimidazole Trihydrochloride
calf thymus DNA
Chondroitin 4-Sulfate
dimethylmethylene blue
Fluorometry
Papain
Pellets, Drug
Proteoglycan
Spectrophotometry
Vision
MC3T3-E1 cells (2 × 104 cells) were seeded on HC300 (n = 5) in 24-well plates and cultured in alpha-minimal essential medium containing 10% fetal bovine serum, streptomycin (100 μm mL−1; FUJIFILM Wako Pure Chemical Corporation), penicillin (100 U mL−1; FUJIFILM Wako Pure Chemical Corporation), and amphotericin B (0.25 μg mL−1; FUJIFILM Wako Pure Chemical Corporation). The culture medium was changed every 3 days. At days 7 and 14 of culture, the cells were washed thrice with phosphate-buffered saline (PBS; FUJIFILM Wako Pure Chemical Corporation) and they were lysed with cell lysis buffer. Then, ALP activity was assessed using an ALP assay kit (FUJIFILM Wako Pure Chemical Corporation) according to the manufacturer’s instructions. Absorbance at 405 nm was measured using a microplate reader (Multiskan FC; Thermo Fisher Scientific, MA, USA).
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alpha minimal essential medium
Amphotericin B
Biological Assay
Buffers
Cells
Culture Media
Fetal Bovine Serum
Penicillins
Phosphates
Saline Solution
Streptomycin
Intracellular ROS levels were measured using the 2,7-dichlorodihydro-fluorescein diacetate (DCFDA) assay, which was applied to a cellular ROS assay kit (ab113851, Abcam) based on the principle of analyzing the byproducts of cellular oxidative metabolism. A549 cells were in 96 well plates at a density of 2.5 × 104 cells/well in triplicate. The cell culture medium was discarded, and the cells were washed with 100 μl pre-warmed 1 × buffer (ab113851, Abcam). The cells were then incubated with 20 μM DCFDA solution (ab113851, Abcam) at 37 °C for 45 min in the dark. After removing the buffer, 100 μl of the prepared PMs were exposed for 1 h, 3 h, and 6 h. Fluorescence was measured using a microplate reader (SpectraMax i3x, Molecular Devices) at Ex/Em = 485/535 nm.
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A549 Cells
Biological Assay
Buffers
Cell Culture Techniques
Cell Respiration
Cells
Culture Media
diacetyldichlorofluorescein
diacetylfluorescein
Fluorescence
Medical Devices
PMS-100
Protoplasm
The DCFDA Cellular ROS Detection Assay kit (Abcam, Cambridge, UK) was used to detect the levels of Reactive Oxygen Species (ROS) in the cells. KG-1 cells were incubated with the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and then plated in duplicates in a 96-well plate and treated with either increasing concentrations of ASEE (19–114 μg/mL), or with a combination of 75 μM of Tert-Butyl Hydrogen Peroxide (TBHP) with increasing concentrations of the ASEE (control cells were treated with RPMI media). TBHP is a potent ROS inducer inside the cells, so it was used as a positive control. H2DCFDA (the chemically reduced form of fluorescein) acts as an indicator for ROS in cells upon its oxidative conversion to the highly fluorescent 2′,7′-dichlorofluorescein (DCF), which was quantified by fluorescent spectroscopy on the Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
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2',7'-dichlorodihydrofluorescein diacetate
Biological Assay
Cells
diacetyldichlorofluorescein
Fluorescein
Helper-Inducer T-Lymphocyte
Reactive Oxygen Species
SERPINA3 protein, human
Spectrum Analysis
tert-Butylhydroperoxide
ROS were measured using the OxiSelect Intracellular ROS Assay Kit (Cell Biolabs, Atlanta, GA, USA) based on the manufacturer's instructions. Briefly, 5 × 104 cells were plated onto a 96-well plate and incubated at 37°C overnight. A specific ROS-detecting fluorescent dye, 2′,7′,-dichlorodihydrofluorescein diacetate (DCFH-DA) was added for 1 h. Cells were washed with PBS twice, medium and 2X cell lysis buffer were added, and fluorescence was measured using the BioTek Synergy HT (Em: 480 nm/Ex: 530 nm). Fluorescence intensity was normalized using the MTT assay.
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Biological Assay
Buffers
Cells
Fluorescence
Fluorescent Dyes
Protoplasm
To prepare 3D tumor spheroids, PANC‐1 and BxPC‐3 cells were seeded in the Corning 96 Well Ultra Low Attachment Microplate at a density of 2 × 103 cells per well. After 5 days, the compacted tumor spheroids were treated with different drug formulations. On day 7, the morphology and number of the spheroids were recorded under an inverted microscope.
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Cells
Microscopy
Neoplasms
The cochleae were dissected from the mouse and homogenized on ice. Because of limited tissue, we combined 10-15 mice cochleae for the study. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen), and cDNA was generated using the RT2 First Strand Kit (Qiagen). cDNA was combined with RT2 SYBR Green Master Mix (Qiagen), specific qRT-PCR primers, and qRT-PCR analysis was run using the ViiaTM 7 Real-Time PCR System (ABI). Primer efficiencies were determined by standard dilution curve analysis. Three separate samples were used from 10 animals for each group. The experiments from each sample were performed in triplicate, and average cycle threshold (Ct) values were normalized to Gapdh expression. ΔΔCt values were determined relative to Cldn9+/+ cochlear samples. Fold change was defined as 2(−ΔΔCt). Primers used include Gapdh (SA Biosciences) and Cldn9 (ThermoFisher).
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Animals
Cochlea
DNA, Complementary
GAPDH protein, human
Mice, Laboratory
Oligonucleotide Primers
SYBR Green I
Technique, Dilution
Tissues
The extracted nucleic acids (from a Roche MagNApure compact automated system) were used for 1-step real-time PCR for RSV detection, using a Qiagen 1-step RT-PCR kit. Real-time RT-PCR assays were performed with primers and probes for RSV, using the CDC protocol [11 ]. All assays were run with positive controls (RSV-RNA), negative controls (nuclease-free water), and internal positive controls (RNAseP) for quality-control purposes.
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Biological Assay
Nucleic Acids
Oligonucleotide Primers
Real-Time Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
Cell cultures and Western blots were performed as described previously [40 (link), 41 (link)]. Human embryonic kidney (HEK) 293T cells obtained from CCTCC were recently tested for mycoplasma contamination but not authenticated. Briefly, HEK293T cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Hyclone). Cells of about 70%-80% confluence were transfected using lipofectamine 8000 (Beyotime) according to the manufacturer’s instructions. If shRNA was used, the ratio of shRNA to expression constructs is 1:1. Forty-eight hours later, cells were lysed in ice-cold radioimmunoprecipitation (RIPA) buffer containing protease inhibitor cocktail (Thermo Scientific). Samples were then loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to membrane, the samples were then probed with rabbit anti-FLAG antibody (1:1000; #14793, CST) or rabbit anti-GAPDH antibody (1:1000; LF206, Epizyme), developed with species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000; LF102, Epizyme) and visualized with enhanced chemiluminescence (Epizyme). For all Western blots, three cultures for each group were used.
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Antibodies
Antibodies, Anti-Idiotypic
Buffers
Cell Culture Techniques
Cells
Chemiluminescence
Common Cold
Embryo
Fetal Bovine Serum
GAPDH protein, human
HEK293 Cells
Homo sapiens
Horseradish Peroxidase
Kidney
Lipofectamine
Mycoplasma
Protease Inhibitors
Rabbits
Radioimmunoprecipitation Assay
SDS-PAGE
Short Hairpin RNA
Tissue, Membrane
Western Blot
Cells were sorted as described in “Tissue Preparation and Flow Cytometry” and immediately lysed with RLT Buffer from the Qiagen RNeasy Plus Mini kit or RNA extraction buffer from PicoPure RNA isolation kit (Arcturus Bioscience, Inc.). Cell lysates were stored at −80°C until RNA was extracted. RNA isolations were performed using the Qiagen RNeasy Plus Mini kit or PicoPure RNA isolation kit. RNA quality and quantity were measured using the Agilent Technologies High Sensitivity RNA ScreenTape System. SMART-Seq v4 Ultra Low Input RNA kit (Clontech Laboratories, Inc.) was used for full-length cDNA synthesis, and the Nextera XT DNA sample preparation kit (Illumina Inc.) was used for library preparation. DNA libraries were sequenced on an Illumina NextSeq 500 instrument with a target read depth of ∼10 million aligned reads per sample. The pool was denatured and diluted, resulting in a 2.5 pM DNA solution. PhiX control was spiked at 1%, and the pool was sequenced by 1 × 75 cycles using the NextSeq 500 High Output reagent kit (Illumina Inc.).
Anabolism
Buffers
Cells
DNA, Complementary
DNA Library
Flow Cytometry
Hypersensitivity
isolation
Tissues
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