Cell-free supernatants were used to enumerate cytokine protein levels using Enzyme-Linked Immunosorbent Assay (ELISA) assay. All the ELISAs were run according to manufacturer instructions: murine IL-6 (BD), murine IL-12p40 (BD), murine IFN-γ (Mabtech), murine IL-2 (BD), murine IL-17A (Invitrogen), and murine IL-22 (R&D Systems). Plates were washed using a Biotek ELx405 machine and measured using a BioTek ELx808 reader.
Elx808 reader
Manufactured by Agilent Technologies
Sourced in United States
The ELx808 reader is a microplate absorbance reader designed for a variety of applications in life science research and clinical diagnostics. It provides accurate and reliable optical density measurements across multiple wavelengths.
Automatically generated - may contain errors
Lab products found in correlation
Gen5 data analysis software, by Agilent Technologies (2 mentions)
Murine il 2, by BD (1 mentions)
Murine ifn γ, by Mabtech (1 mentions)
Synergy htx reader, by Agilent Technologies (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
Tal test reagent kit, by Xiamen (1 mentions)
Endosafe endochrome k, by Charles River Laboratories (1 mentions)
10 protocols using elx808 reader
1
Quantification of Cytokine Levels by ELISA
2
Colorimetric Assay for Ceruloplasmin
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CP oxidase activity on N,N-dimethyl-p-phenylenediamine substrate in waste fractions I, III, IV1-4 and in purification process intermediates was determined by the EIACPLC—Ceruloplasmin Colorimetric Activity Kit (Invitrogen) at 562 nm using an ELX-808 reader (BioTek). Commercially available research grade CP products (ALX-200-089 ENZO LIFE SCIENCES and C4519 Sigma Aldrich) were used as controls. CP ferroxidase activity on (Fe2+) substrate was determined in purified CP concentrate according to a modified Erel method74 (link) (see below). A calibration curve was generated by diluting CP standard included in the EIACPLC kit Invitrogen (range 148–29.5 mU/ml) with 45 mM Na-acetate, pH 5.8, and commercial CPs used as internal controls. The detection at 595 nm was carried out with a Synergy HTX reader (BioTek). Before analysis kCP lyophilized concentrate samples were dialyzed against saline solution, to avoid buffer interference as attested with citrate buffer.
3
Characterizing Microbial Growth Patterns
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Drop tests were performed with fresh cells resuspended in water and adjusted to the same initial OD600 = 2.0 (Eppendorf Biophotometer). Tenfold serial dilutions were prepared, and 3 μL aliquots of each dilution were spotted on the appropriate media supplemented as indicated in the text. For the growth assays in solid and liquid media with different pH values, the pH was adjusted with tartaric acid or NaOH. Plates were incubated at 30°C for 48 hours and photographed daily with a Nikon Coolpix7000 camera. Each drop test was performed three times and representative results are shown. The growth in liquid media was monitored in 96-well plates at 30°C for 48 h. In the wells, 100 μL of the media (indicated in the text) were inoculated with 2 μL of cell suspension (OD600 = 2). The OD600 was measured using an Elx808 reader (BioTek) at 1-h intervals. Growth curves were obtained with four technical replicates for each strain and condition, and in two independent experiments.
4
Kinetic Turbidimetric Assay for LPS Quantification
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The concentration of LPS in the supernatants of cell lysates was determined by the kinetic turbidimetric assay using the tachypleus amebocyte lysate (TAL) test reagent kit purchased from Xiamen Bioendo Technology Co., Ltd. The supernatant was diluted with endotoxin-free water. The lyophilized endotoxin standard stock was reconstituted and diluted to final concentrations of 10, 1, 0.1, 0.05, and 0.01 EU ml− 1. All samples, standards and negative controls were placed into the wells of a 96-well plate (non-pyrogens) in duplicate. 100 µl TAL reagent was added into each well, and the plate was incubated at 37 °C in a BioTek ELx808 reader (BioTek Instruments, Inc.) for 2 h. The endotoxin level was measured using a kinetic assay program and calculated using Gen5™ data analysis software (BioTek Instruments, Inc.).
5
Quantitative Endotoxin Determination in Plasma
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At 0, 2 and 4 h after KB treatment, the concentration of LPS in the plasma was determined using the LAL test. Briefly, each sample (100 μl) was added into 100 μl quantitative LAL reagents dissolved in LPS-free water and reacted at 37°C for 2 h. The gel clotting formation of the LAL products induced by the existence of non-neutralized LPS was measured through the kinetic turbidimetric assay, in which endodoxin triggers a cascade of enzymatic reactions to activate the clotting enzyme. The formation of the gel clot is proportional to the concentration of endotoxin in the sample. Aliquots (100 μl) of all samples, standards and negative controls were seeded into a 96-well plate (non-pyrogens) and incubated at 37°C in a BioTek ELx808 reader (BioTek Instruments, Inc., Winooski, VT, USA). for 10 min. Following incubation, ~100 μl quantitative LAL reagents, dissolved in LPS-free water, rotate up and down until the solution turned clear prior to use, was added to each well (Chinese Horsehoe Crab Reagent Manufacturery Co., Ltd., Xiamen, China). Following gentle vibration for 10 sec, the absorbance at 630 nm was measured and readings were repeated every 30 sec for 2 h. The results were calculated using Gen5™ data analysis software (BioTek Instruments, Inc.).
6
Quantitative IL-6 Immunoassay Protocol
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The DIA source IL-6-EASIA kit was used (Engvall and Perlmann, 1971 (link)). The assay uses monoclonal antibodies (MAbs) directed against distinct epitopes of IL-6. Calibrators and samples react with the capture monoclonal antibody (MAb 1) coated on microtiter well and with a monoclonal antibody (MAb 2) labeled with horseradish peroxidase (HRP). After an incubation period to allow the formation of a sandwich: coated MAb 1 – human IL-6 – MAb 2 – HRP, the microtiter plate is washed to remove unbound enzyme labeled antibody. Bound enzyme labeled antibody is measured through a chromogenic reaction. Chromogenic solution (TMB) is added and incubated. The reaction is stopped with the addition of stop solution and the microtiter plate is then read at the appropriate wavelength. The amount of substrate turnover is determined colorimetrically by measuring the absorbance, which is proportional to the IL-6 concentration. The plates were first read at 450 nm against a reference filter set at 650 nm (or 630 nm) by use of BioTek ELx808 reader. A second reading was performed at 490 nm against the same reference filter. The software automatically drove the reader and integrated both readings into a polychromatic model. Data were statistically analyzed and represented as mean ± sd for IL-6.
7
Kinetic Chromogenic LAL Assay Protocol
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The kinetic chromogenic LAL assay (Endosafe Endochrome-K™, Charles River Laboratories) was used according to the manufacturer’s instructions. In the following manuscript, LAL is referred to as Endosafe Endochrome-K™. The absorption at 405 nm was measured using an Elx808 reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany). All samples were measured in duplicate and average values were used. Standard curves were fitted using a linear regression model. The detection limit of the assay was 0.005 EU/mL. In order to control test interference, positive product controls (PPC) according to manufacturers’ instructions were performed. Data of each run were regarded as valid data only if the following acceptance criteria were met:
The temperature during the measurement must be 37 ± 1.0 °C.
Fit of the standard curve: r ≤ −0.980.
Linear regression: Slope must be between −0.400 and −0.100.
Linear regression: Y Intercept has to be between 2.500 and 3.500.
The mean onset-time of the blank must be higher than the mean onset-time of the lowest standard concentration.
The Coefficient of Variation (CV) of all replicates must be ≤10%.
The PPC must be between 50% and 200%. Therefore, 0.5 EU/mL Control Standard Endotoxin (CSE) was spiked into the diluted and undiluted sample, respectively.
8
Erythrocyte Lipid Peroxidation Assay
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Lipid peroxidation in erythrocyte membranes is determined according to the method of Stocks and Dormandy (1971) [38 (link)]. Lipid peroxidation is analyzed by measuring of formation of thiobarbituric acid reactive substances (TBARS). The absorbance is determined colorimetrically using BioTek ELx808 reader (Winooski VT, USA) at the wavelength of 532 nm. Lipid peroxidation is expressed in absorbance units of TBARS products and is shown as a percentage of control.
9
Erythrocyte Osmotic Resistance Assay
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The osmotic resistance (fragility) was determined by the method of Dacia and Lewis (1975) [39 ]. A small number of erythrocytes are placed in a NaCl solution at a concentration of 0.2 to 0.9%. Osmotic resistance is determined by measuring the hemoglobin released from erythrocytes by the colorimetric method using the BioTek ELx808 reader (Winooski VT, USA) at λ = 540 nm. Osmotic resistance is assessed on the basis of the hemolysis curve shift, in the graph of percentage of hemolysis vs. NaCl concentration. Before performing the assay, RBCs were incubated with test compounds for 3 h. The exact procedure for performing the experiment is described in the publication by Maćczak et al. (2017) [21 (link)].
10
Yeast Adherence Quantification by XTT
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Adherence of yeast strains was determined with the method of tetrazolium salt reduction (XTT; Sigma-Aldrich) accordingly to Jin et al. (2004 ). The experiment was conducted on 96-well titration plates (Sarstedt). Before being used, the plates were coated with 3% BSA (100 μL/well) and left overnight at a temp. of 4 °C. Next, the excess of bovine serum albumin was decanted, and the plates were double-rinsed with HEPES (pH 7.0). The suspension of yeast strains (100 μL) was introduced to wells and incubated at a temp. of 22 °C. To determine cell adherence degree (after 2, 6, 12, 24, and 48 h of incubation), the excess of culture was removed and the wells were double-rinsed with PBS. Afterwards, 100 μL of the reaction mixture containing 1580 μL PBS, 200 μL XTT, and 20 μL menadione (2-methyl-1,4-naphthoquinone; Sigma-Aldrich) was added accordingly to modified method of Ramage et al. (2001 (link)). The plates were incubated at 22 °C/2 h, and then absorbance of the supernatant was measured at λ = 490 nm (Antachopoulos et al. 2006 (link)). Measurements were made with the Elx808 reader (BioTek Instruments) (Balcazar et al. 2008 ).
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