At baseline, the serum NETs were assessed using a neutrophil elastase ELISA as previously described [Citation20]. Briefly, high binding 96 well plates were coated with mouse anti-human neutrophil elastase (Calbiochem, Darmstadt, Germany) diluted 1:2000 in coating buffer from the cell death detection ELISA kit (Roche, Basil, Switzerland) and incubated at 4 °C, overnight. Plates were washed and then blocked with 1% bovine serum albumin (BSA) in PBS for 6 h at room temperature. Serum samples (1:10 in 1% BSA) were added to the plates and incubated at 4 °C, overnight. The following day, the plates were washed and then incubated with the anti-human DNA-POD antibody (1:10) from the cell death detection ELISA kit (Roche) in incubation buffer, for 1 h at room temperature. Afterwards, 5 washes, TMB substrate was added to each well (Thermofisher Scientific, Walthem, MA, USA), and the reaction was finished by adding stop solution. The plates were read at 450 nm and the optic density index was calculated as previously described [Citation20] in a Tecan Sunrise-RC/ST Evolyzer Plate Reader.
Cell death detection elisa kit
Manufactured by Roche
Sourced in United States, Germany, Switzerland, Spain, France
The Cell Death Detection ELISA kit is a quantitative in vitro assay for the detection and quantification of DNA fragmentation, a hallmark of apoptosis. The kit uses an enzyme-linked immunosorbent assay (ELISA) technique to detect and measure cytoplasmic histone-associated DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
High binding eia ria 96 well plate, by Corning (8 mentions)
Anti human mpo antibody, by Bio-Rad (8 mentions)
Microplate reader, by Agilent Technologies (5 mentions)
Stop reagent, by Merck Group (5 mentions)
Tmb substrate, by Merck Group (5 mentions)
Camptothecin, by Merck Group (5 mentions)
Spectramax m5, by Molecular Devices (4 mentions)
Synergy ht multi mode microplate reader, by Agilent Technologies (4 mentions)
Anti dna pod clone mca 33, by Roche (4 mentions)
Mouse anti human mpo clone 4a4, by Bio-Rad (4 mentions)
Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (4 mentions)
Facscalibur, by BD (3 mentions)
Tetramethylbenzidine tmb substrate, by Thermo Fisher Scientific (3 mentions)
Cytation 5 cell imaging multi mode reader, by Agilent Technologies (3 mentions)
Tmb substrate, by Thermo Fisher Scientific (3 mentions)
Ultra multifunctional microplate reader, by Tecan (3 mentions)
Mtt assay, by Roche (3 mentions)
Infinite 200 pro, by Tecan (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Ab5103, by Abcam (3 mentions)
275 protocols using cell death detection elisa kit
1
Quantifying Serum Neutrophil Extracellular Traps
2
Quantifying Serum Neutrophil Extracellular Traps
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As previously described, serum NETs levels were assessed using a neutrophil elastase enzyme-linked immunosorbent assay (ELISA) (17 (link)) at baseline and 3 months after recruitment. Briefly, mouse anti-human neutrophil elastase (Calbiochem, Darmstadt, Germany) was diluted 1:2000 in coating buffer from the cell death detection ELISA kit (Roche, Basil, Switzerland) and incubated in high-binding 96-well plates overnight at 4 °C to generate mouse anti-human neutrophil elastase-coated plates. Plates were washed three times with PBS/Tween20 and then blocked with 1% bovine serum albumin (BSA) in PBS for 6 h at room temperature. Afterward, serum samples (1:10 in 1% BSA) were added to the plates and incubated overnight at 4 °C. Following three washes with PBS/Tween20 solution, the samples were incubated with anti-human DNA-peroxidase antibody (1:10 dilution in incubation buffer) from the cell death detection ELISA kit (Roche) for 1 h at room temperature. Next, the plates were washed five times with PBS/Tween20 before adding TMB substrate (Thermofisher Scientific, Waltham, MA, USA), followed by a stop solution. The plates were read at 450 nm, and the optic density index was calculated as previously described (17 (link)) in a Sunrise-RC/ST Evolyzer Plate Reader (Tecan Life Sciences, Männendorf, Switzerland).
3
Quantifying Plasma Neutrophil Extracellular Traps
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The amount of plasma NETs was addressed by ELISA, as previously described (18 (link)). Briefly, high binding 96 well plates were coated overnight at 4°C with mouse anti human neutrophil elastase (NE) 1:2000 (Cat 481001, Calbiochem, Darmstadt, Germany) in coating buffer from the cell death detection ELISA kit (Roche, Basilea, Switzerland). We washed the plates three times with PBS/Tween20 and blocked the non-specific binding sites with 1% bovine serum albumin (BSA) in PBS for 6 hrs at room temperature for the detection of DNA-NE complexes as previously described (18 (link)). The plasma samples were diluted 1:10 in 1% BSA and incubated overnight at 4°C. After washing three times with PBS/Tween 20, we incubated the plates with the anti-human DNA-POD antibody from the cell death detection ELISA kit (Roche, Basilea, Switzerland). We washed the plate five times with PBS/Tween 20 and applied the TMB substrate (Thermofisher Scientific, Massachusetts, USA). The plate was read at 450 nm after applying stop solution and the optic density index (ODI) was calculated as previously described (18 (link)).
4
Quantifying NETs and MPO/DNA Complexes
5
Quantifying Serum MPO-DNA Complexes in SLE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum MPO-DNA complex levels of SLE patients and HCs were measured as previously reported18 (link). In brief, high-binding 96-well microplates (Corning) were incubated overnight at 4 °C with a mouse anti-human MPO antibody (clone 4A4; Bio-Rad) diluted in coating buffer (Cell Death Detection ELISA kit; Roche). Following blocking with 1% bovine serum albumin (cat# A2153; Sigma) in phosphate-buffered saline (PBS), plates were incubated at room temperature with 10% human serum in blocking buffer, washed, and then 10 × anti-DNA-POD (Clone MCA-33, Cell Death Detection ELISA kit; Roche) in blocking buffer was added. After the incubation, TMB substrate (KPL) was added, and absorbance was measured at 450 nm after the addition of the stop reagent (Wako). Serum MPO-DNA complex “high” patients were defined as those whose optical density values were two standard deviations higher the mean of HCs.
6
Molecular Mechanisms of CCN5-Mediated Epithelial-Mesenchymal Transition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, Aprotinin, PMSF, Leupeptin, trypsin EDTA solution, sodium pyruvate, 17β-estradiol (E2), leptin and β-actin monoclonal antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Human recombinant CCN5 protein (hrCCN5) was purchased from PeproTech (Rocky Hill, NJ, USA). Anti- E-cadherin and anti-vimentin antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. Anti-WISP-2/CCN5 rabbit polyclonal antibody and Anti-Snail antibody were purchased from Abcam (Cambridge, MA, USA). Super Signal ULTRA chemiluminescent substrates were obtained from Pierce, Rockford, IL. Cell-death detection ELISA kits were purchased from Roche Diagnostic (Indianapolis, IN). The authentication certificates for all these chemicals, drugs and antibodies were provided by these companies. The fresh working solutions of the chemicals and drugs were prepared once a month to guarantee effectivity.
7
Evaluating Proliferation and Apoptosis of hRPE Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell proliferation and cell death enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate the effect of the A/G substrate on the proliferation or unfavorable apoptosis of hRPE cells in culture. As mentioned previously, A/G substrates were placed in each well of a 96-well microplate. At Passage 4, cells were seeded (1 × 104 cells) on A/G substrate in the presence of 200 µL DMEM/F12 culture media without serum or supplemented with either 30% HAF or 10% FBS and incubated at 37 °C in a 5% CO2 incubator for 2 days. Cultures on polystyrene substrates that underwent similar treatments were assessed as controls. Cell proliferation and cell death assays were performed according to the manufacturer’s instructions (cell proliferation ELISA, BrdU colorimetric, and cell death detection ELISA kits, Roche, Grenzach-Wyhlen, Germany). The results are presented as the means of three independent experiments performed at least in triplicate.
8
Apoptotic Cell Death Determination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptotic cell death was determined using cell death detection ELISA kits (Roche Diagnostic, Indianapolis, IN, USA) as we previously described [70 (link)]. Briefly, untreated and treated cells were harvested, lysed with lysis buffer, and the protein in the cytoplasmic supernatant was measured. Approximately 20 μL from the supernatant was added in the streptavidin-coated microplate and incubated with 80 μL of buffer mixture containing anti-histone-biotin and anti-DNA–peroxidase (POD) for 2 h with continuous shaking at room temperature. Microplates were washed with incubation buffer and the ABTS [2,2¢-azino-di-[3-ethylbenzthiazoline-6-sulfonic acid)) chromogen substrate was added to get a color. The reaction between POD and ABTS was photometrically determined using a microplate reader at 405 nm.
9
Cell Proliferation and Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell number was measured using trypan blue exclusion on a cell counter device Cellometer Auto T4 Bright Field Cell Counter (Nexcelom Bioscience LLC, Lawrence, MA, USA). Cell viability/proliferation was measured by counting the cell numbers in using Crystal Violet staining followed by the measurement of absorbance at 600 nm using SOFTmaxPRO. Apoptosis was assayed using cell-death detection ELISA kits (Roche Diagnostic Corporation, Indianapolis, IN, USA).
10
Serum Histone Quantification by ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum histone levels were measured by Cell Death Detection ELISA kits (Roche) according to the manufacturer’s instructions. Briefly, reconstitute the Anti-Histone Biotin and Anti-DNA POD in 450 μl double distilled water and dissolve ABTS tablets in 15 ml Substrate Buffer. The Immunoreagent is prepared by mixing of 1/20 volume Anti-DNA-POD and 1/20 volume Anti-histone-biotin with 18/20 volumes Incubation Buffer. Add to each well 20 μl serum sample and 80 μl of the Immunoreagent. Incubate on a MP shaker under gently shaking (300 rpm) for 2 h at room tempreture. Remove the solution thoroughly and rinse each well 3× with 250 μl Incubation Buffer. Pipette to each well 100 μl ABTS solution. Incubate on a plate shaker at 250 rpm for 15 min. Pipette to each well 100 μl ABTS Stop Solution. Measure and analyze at 405 nm against 490 nm wavelength.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!