The concentrations or activity of amylase (ab102523; Abcam), MPO (EMMPO; Thermo Fisher Scientific), trypsin (ab102531; Abcam), 8-hydroxy-2-deoxyguanosine (K4160; BioVision, Milpitas, CA), HMGB1 (326054329; Sino-Test Corporation, Tokyo, Japan), LDH (ab102526; Abcam), cleaved-caspase3 (DYC835-2; R&D Systems, Minneapolis, MN), p-MLKL (PEL-MLKL-S345-1; RayBiotech Life, Norcross, GA), MDA (ab118970; Abcam), iron (MAK025; Sigma-Aldrich), GSH (CS0260; Sigma-Aldrich), CoQ10 (MBS7233016; MyBioSource, San Diego, CA), tumor necrosis factor (BMS607-3; Thermo Fisher Scientific), IL6 (BMS603-2; Thermo Fisher Scientific), and IL1B (BMS6002; Thermo Fisher Scientific) in the indicated samples were measured using enzyme-linked immunosorbent assay kits according to the manufacturer's guidelines. Data were normalized to protein or DNA concentration.
8 hydroxy 2 deoxy guanosine
Manufactured by Abcam
Sourced in United States
8-hydroxy-2-deoxy guanosine is a nucleoside derivative formed by the oxidation of deoxyguanosine. It is commonly used as a biomarker to measure oxidative stress and DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
Synergy h1 hybrid multi mode reader, by Agilent Technologies (2 mentions)
Rabbit anti 8 ohdg, by Abcam (2 mentions)
Ab102531, by Abcam (1 mentions)
Bms607 3, by Thermo Fisher Scientific (1 mentions)
Coq10, by MyBioSource (1 mentions)
Bms6002, by Thermo Fisher Scientific (1 mentions)
Ab118970, by Abcam (1 mentions)
Ab102523, by Abcam (1 mentions)
Mak025, by Merck Group (1 mentions)
Dyc835 2, by R&D Systems (1 mentions)
Bms603 2, by Thermo Fisher Scientific (1 mentions)
Ab102526, by Abcam (1 mentions)
Ab201734, by Abcam (1 mentions)
F4 80, by Bio-Rad (1 mentions)
Cyclin d1, by Agilent Technologies (1 mentions)
P smad3, by Abcam (1 mentions)
Pparγ, by Cell Signaling Technology (1 mentions)
P19arf, by Santa Cruz Biotechnology (1 mentions)
γh2ax ser139, by Merck Group (1 mentions)
Zen 3, by Zeiss (1 mentions)
7 protocols using 8 hydroxy 2 deoxy guanosine
1
Comprehensive Biochemical Analysis of Samples
2
Quantifying 8-OHdG in Cell Supernatant
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Determination of 8-OHdG content in cellular supernatant was assed using an in-house competition ELISA. Clear 96-well plates were coated with 8-Hydroxy-2’-deoxyguanosine (BioVision, Milpitas, CA) overnight at 4°C. Plates were then washed three times with PBS. Cell supernatant in PBS or standard 8-OHdG standard curve dilutions were added to the appropriate wells. Rabbit anti-8-OHdG (Abcam, Cambridge, MA) was subsequently added at a 1:2,500 final dilution in PBS and the plate was incubated at room temperature with shaking for 2 h. Following three washes with PBS, anti-rabbit HRP antibody was added at a 1:5,000 dilution and the plate was incubated at room temperature with shaking for 1 h. Immediately following five rapid PBS washes, 3.7mM o-phenylenediamine in 0.05 M phosphate-citrate buffer containing 0.03% sodium perborate was added and the plate was incubated at room temperature protected from light for 30 min. Absorbance at 405 nm was then measured on a Synergy H1 Hybrid Multi-Mode Reader (BioTek).
3
Quantifying 8-OHdG in Cell Supernatant
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Determination of 8-OHdG content in cellular supernatant was assed using an in-house competition ELISA. Clear 96-well plates were coated with 8-Hydroxy-2'-deoxyguanosine (BioVision, Milpitas, CA) overnight at 4°C. Plates were then washed three times with PBS. Cell supernatant in PBS or standard 8-OHdG standard curve dilutions were added to the appropriate wells. Rabbit anti-8-OHdG (Abcam, Cambridge, MA) was subsequently added at a 1:2500 final dilution in PBS and the plate was incubated at room temperature with shaking for 2 hours. Following three washes with PBS, anti-rabbit HRP antibody was added at a 1:5000 dilution and the plate was incubated at room temperature with shaking for 1 hour. Immediately following five rapid PBS washes, 3.7mM o-phenylenediamine in 0.05 M phosphate-citrate buffer containing 0.03% sodium perborate was added and the plate was incubated at room temperature protected from light for 30 minutes. Absorbance at 405 nm was then measured on a Synergy H1 Hybrid Multi-Mode Reader (BioTek).
4
Oxidative Stress Markers in Aging
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Oxysterols as markers of oxidative stress and 8-hydroxy-2-deoxy guanosine as a marker of oxidative DNA damage were analyzed in a set of randomly chosen samples of plasma collected from frail, pre-frail, non-frail, and young individuals. 7-Keto-cholesterol (7KC), 7β-hydroxycholesterol (7βOHC), 5α6α-epoxycholesterol (5α,6αEC), 5β6β-epoxycholsterol (5β,6βEC), and 3β5α6β-3OH-cholesterol (triol) (3β,5α,6β-3OHC) were quantified using isotope dilution mass spectrometry (28 (link)) as cholesterol oxidation and cholesterol autoxidation products resulting from oxidative stress (29–31 (link)). 8-hydroxy-2-deoxy guanosine (8-OH-dG) was analyzed in duplicate by enzyme-linked immunosorbent assay (ab201734, 8-hydroxy-2-deoxy guanosine, ELISA kit, Abcam, Cambridge, UK), according to literature (32 (link)) and the manufacturer’s instructions.
5
Oxidative Stress Biomarkers Assessment
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Serum concentrations of Cu/Zn SOD and 8-hydroxy-2-deoxyguanosine were determined with ELISA kits (Cu/Zn SOD: Northwest Life Sciences Inc., Vancouver, WA, USA; 8-hydroxy-2-deoxyguanosine: Abcam, Cambridge, UK), according to the instructions of the manufacturer. Absorbance was read on a spectrophotometer (Thermo Luminoskan Ascent, Waltham MA, USA) at 450 nm (Cu/Zn SOD) and 410 (8-hydroxy-2-deoxyguanosine).
6
Histological Analysis of Tissue Samples
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Tissues were fixed in 10% buffered formalin, embedded in paraffin wax and sectioned at 5 mm. For histological examination sections were stained with hematoxylin and eosin, according to standard procedures as previously described [62 ]. CC3 Cleaved Caspase 3 Asp175 (Cell Signaling Technology 9661), CC10 (Santa Cruz Biotechnology sc-9772), CD4 (Cell Signaling Technology 25229, prosurfactant protein C (millipore AB3786), p21 (291 H/B5, homemade), γH2AX Ser 139 (Millipore 05-636), PPERK Thr202/Tyr204 (Cell Signaling Tehcnology 9378), Ki67 (Cell Signaling 12202), MPO (Dako A0398), F4-80 (ABD Serotec MCA497), p16 (33B, homemade), p19 ARF (sc-32748 Santa Cruz), pRb (ser807/811, #9308, Cell Signaling), pSMAD3 (ser423/425 ab52903, Abcam), pSTAT3 (tyr705, #9145 Cell Signaling), p53 (POE316A, homemade), cyclin D1 (M3635, Dako), c-MYC (ab32072, Abcam), PPARγ (Cell Signaling, #2435), HIF1α (Cell Signaling, #36169), 8-hydroxy-2’-deoxyguanosine (Abcam, ab48508), 4-hydroxy-2-nonenal (Alpha Diagnostic, HNE11-S) antibodies were used for immunohistochemistry in tissue sections. Pictures were taken using Olympus AX70 microscope. The percentage of positive cells was identified by eye and the areas were calculated by ImageJ and Zen 3.1 (Zeiss) softwares.
7
Biomarkers of COVID-19 Severity
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We assessed by ELISA, following the provider instructions, the plasma levels of TRIM63 (MyBioSource, California, USA) and atrogin-1 (MyBioSource, California, USA) as markers of muscle atrophy. The serum concentrations of HIF-1α (Thermofisher Scientific, Massachusetts, USA) and 8-hydroxy 2 deoxyguanosine (Abcam, Cambridge, UK) to address tissue hypoxia and oxidative stress, and the plasma levels of TRIM21 (MyBioSource, California, USA) as part of the antiviral innate immune response were measured by a commercial ELISA as well.
Noteworthy, all the samples were processed by investigators blinded to the COVID-19 severity and outcome of each included subject.
Noteworthy, all the samples were processed by investigators blinded to the COVID-19 severity and outcome of each included subject.
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