Cell viability was measured using the colorimetric CyQuant cell proliferation assay (Invitrogen), following the manufacturer’s instructions. Absorbance was analyzed at 480–520 nm, using 50,000 cells harvested from whole conventional and silk-VM organoids. Each organoid was analyzed in 6 replicates (i.e., 300 000 cells from each organoid) using Biochrom Asys Expert 96 Microplate Reader (Biochrom). Apoptosis was detected via flow cytometry after staining using the Alexa Fluor 647 Annexin V conjugate (BD Pharmingen, #A23204) and Click-iT plus Tunel assay (Invitrogen #C10617) according to the manufacturer’s instructions.
Asys expert 96 microplate reader
Manufactured by Harvard Bioscience
Sourced in United Kingdom
The Asys Expert 96 Microplate Reader is a compact and versatile instrument designed for absorbance measurements in 96-well microplates. It is capable of reading a wide range of wavelengths, from 200 to 1000 nm, enabling a variety of assays and applications.
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Lab products found in correlation
Cyquant cell proliferation assay, by Thermo Fisher Scientific (1 mentions)
Click it plus tunel assay, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Pbs tween 20, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Skim milk, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Cytotoxicity detection kit, by Roche (1 mentions)
6 protocols using asys expert 96 microplate reader
1
Organoid Cell Viability and Apoptosis
2
Quantitative Western Blot Analysis
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Briefly, protein concentrations were measured using Bradford Bio-Rad protein assay according to the manufacturer’s protocol (Bio-Rad, USA). The absorbance measurement was performed following manufacturer’s protocol (Biochrom Asys Expert 96 micro plate reader, Cambridge, UK). 10 μg of proteins were loaded on 4–20% Mini-Protean TGX Precast Gels (Bio-Rad, USA) then transferred to nitrocellulose membranes (Bio-Rad) using Trans-Blot Turbo System (Bio-Rad). Membranes were then blocked with 5% skim milk (Sigma-Aldrich) diluted in PBS-Tween 20 (Sigma-Aldrich). After blocking, the membranes were incubated with the primary antibodies listed in previous section and BMP7 antibody (Abcam; data not shown) at 4 °C, overnight. Incubation with primary monoclonal anti-ß-Actin antibody (1:10,000; Sigma-Aldrich) was performed for 20 minutes only. Membranes were incubated with peroxidase-conjugated secondary antibody (Vector Labs) and blots were developed using Clarity Western ECL Substrate (Bio-Rad) and the protein levels were normalized to actin.
3
Serodiagnosis of Lyme Disease by ELISA
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In the IgM and IgG classes, sera from the experimental and control groups were subjected to the anti-Borrelia ELISA screening test by EUROIMMUN (Lübeck, Germany). A complete panel of B. burgdorferi sensu stricto, B. afzelii and B. garinii antigens was used to evaluate the IgM class. In contrast, the full panel of B. burgdorferi sensu stricto, B. afzelii, B. garinii antigens and the recombinant protein VIsE of B. burgdorferi were used to assess the IgG class. According to the EUCALB requirements for interpreting results in LD diagnosis, as well as recommendations of the test manufacturer, sera with a value of ≥22 RU/mL were considered positive, those ranging between 16 and 22 RU/mL were considered borderline and those <16 RU/mL were regarded as negative [6 (link),23 (link)]. Results were read at 450 nm using a Biochrom Asys Expert 96 Microplate Reader (Biochrom Ltd., Cambridge, UK) and MicroWin 2000 S.C. Reader software (Biogenet, Jozefow, Poland).
4
HCV Serum Antibody ELISA Assay
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The purified recombinant protein was used as the antigen coating the ELISA strips in order to test sera in an ELISA format. The wells of microplates were coated with 100 μl of the antigen (5 μg/ml) in 100 mM sodium carbonate buffer pH 9.6, at 4 °C overnight. Next day, the wells were washed three times with TBST and blocked with 200 μl of 5% BSA in TBS for 1 hr at 37°C. After three times washing with wash buffer, 100 μl of diluted serum (1:1000) was added in duplicate to the wells and was incubated for 30 min at 37°C. The wells were washed three times and subsequently incubated with 100 μl of HRP-conjugated goat anti-human IgG, diluted 1:1000 for 30 min at 37°C. Washing the wells were performed four times, then 100 μl of the TMB substrate was added to each well and incubated for 20 min at 37°C, while microplates were coverslipped with foil. The reaction was stopped by adding 50 μl of 0.2 M H2SO4 and absorbance of the resulting yellow color was measured at 450 nm with the Biochrom Asys Expert 96 microplate reader.
50 HCV positive sera, from different HCV subtypes (1a, 1b, and 3a), and 50 HCV negative sera were used in ELISA test
50 HCV positive sera, from different HCV subtypes (1a, 1b, and 3a), and 50 HCV negative sera were used in ELISA test
5
Mitochondrial Activity Measurement via XTT
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XTT assay was performed to measure mitochondrial activity (mitochondrial dehydrogenase) in living cells using XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide salt) (Sigma-Aldrich, Sweden). The assay was performed following the manufacturer’s protocol in a 96-well plate (Biochrom Asys Expert 96 micro plate reader, Cambridge, UK) (suppl. Fig. 5c, online resource 5).
6
Evaluating Irisin's Impact on Cell Dynamics
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The lactate dehydrogenase activity was evaluated in the culture medium collected at days 3, 7, 14 and 21 with a cytotoxicity detection kit (Roche Diagnostics, Mannheim, Germany)
according to the manufacturer's instructions. In short, a quantity of 50 μl of sample was added to 50 μl of the kit reaction mixture, and incubated for 30 min in the dark at room temperature.
The absorbance of the samples was measured at 492 nm in a plate reader (Biochrom Asys Expert 96 Microplate Reader; Biochrom, Holliston, MA, USA).
The effect of irisin on cell growth and migration were evaluated by cell proliferation and wound-healing assays.
according to the manufacturer's instructions. In short, a quantity of 50 μl of sample was added to 50 μl of the kit reaction mixture, and incubated for 30 min in the dark at room temperature.
The absorbance of the samples was measured at 492 nm in a plate reader (Biochrom Asys Expert 96 Microplate Reader; Biochrom, Holliston, MA, USA).
The effect of irisin on cell growth and migration were evaluated by cell proliferation and wound-healing assays.
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