A chemiluminescence-based assay was used to determine ROS production by neutrophils. Cells were suspended in RPMI-1640 medium without phenol red at a final concentration of 1.5 x 106 cells/ml and were pre-incubated at 37°C in the presence or absence of the priming agent TNF-α (50 ng/ml). Following 10 minutes of incubation, cells were added to a white, clear-bottom, 96-well microtiter plate (PerkinElmer) and stimulated with PMA (150 ng/ml), ultrapure LPS from E. coli (10 µg/ml), PGN from S. aureus (10 µg/ml), CpG oligodeoxynucleotides (3 µM), TNF-α (50 ng/ml) or IL-1β (500 ng/ml) in the presence of 5 mM luminol (Sigma-Aldrich). Kinetic measurements of luminol oxidation were performed during 1.5 hours at 37°C using a Clariostar Monochromator Microplate Reader (BMG Labtech, Orthenberg, Germany). The results were subtracted by values obtained with PMA stimulation in the absence of luminol.
Clariostar monochromator microplate reader
Manufactured by BMG Labtech
Sourced in Germany, United States
The Clariostar Monochromator Microplate Reader is a multi-mode microplate reader that utilizes monochromators to provide wavelength-specific illumination and detection. It is capable of absorbance, fluorescence, and luminescence measurements across a wide range of microplate formats.
Automatically generated - may contain errors
Lab products found in correlation
Luminol, by Merck Group (2 mentions)
Griess reagent system, by Promega (2 mentions)
Luminol, by BMG Labtech (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Ultrasensitive elisa, by Mercodia (1 mentions)
White 96 well microplate, by PerkinElmer (1 mentions)
N formyl methionyl leucyl phenylalanine fmlf, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Tumor necrosis factor α tnf α, by Thermo Fisher Scientific (1 mentions)
Rpmi medium without phenol red, by Thermo Fisher Scientific (1 mentions)
Peptidoglycan pgn from staphylococcus aureus, by Merck Group (1 mentions)
Lipopolysaccharide lps from pseudomonas aeruginosa, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Texas red labeled dextran, by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Pge2 parameter assay kit, by R&D Systems (1 mentions)
Phrodo red zymosan bioparticles, by Thermo Fisher Scientific (1 mentions)
Macsxpress whole blood neutrophil isolation kit, by Miltenyi Biotec (1 mentions)
Labassay creatinine assay, by Fujifilm (1 mentions)
24 protocols using clariostar monochromator microplate reader
1
Chemiluminescence-Based Neutrophil ROS Assay
2
Quantifying Insulin Residue on Catheter Membranes
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Catheter membranes from seven of the eleven subjects were analyzed for insulin residue (Fig. 1 ). The frozen membranes were thawed at room temperature. 50 µL phosphate buffered saline (PBS) (Medicago AB, Uppsala, Sweden) with 0.05% Tween 20 (Bio-Rad, California, USA) was added to each vial. After an incubation overnight at 4 °C, the vials were placed on a plate shaker with occasional mixing at room temperature for four hours to extract the adsorbed proteins from the membranes. This method has previously been used for detection of proteins adsorbed to the catheter membrane in surgically treated intracerebral hemorrhage patients19 (link). Insulin concentration was then measured using ultrasensitive ELISA (Mercodia AB, Uppsala, Sweden) and a Clariostar Monochromator Microplate reader with a 450 nm filter (BMG Labtech, Ortenberg, Germany).
3
Quantifying Neutrophil ROS Production
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To quantify ROS production, PMNs were diluted in RPMI-1640 medium without phenol red (3 × 106 c/mL) and first primed with TNF-α (50 ng/mL) for 10 min at 37 °C. We then transferred the cells to a white 96-well microplate (PerkinElmer, Waltham, MA, USA) and added PMA (30 nM), IL-1β (500 ng/mL), LPS (10 µg/mL), PGN (10 µg/mL) or fMLF (10−7 M). Buffer was used to simulate control conditions whereas PMA was used as positive control. Each condition was included in duplicate. The cells were supplemented with 10 mM luminol. The luminol oxidation was monitored for 3 h, measuring the light signal every minute (Clariostar Monochromator Microplate Reader, BMG Labtech, Ortenberg, Germany). During this period, the temperature was kept constant at 37 °C. ROS production by PMNs was assessed by calculating the difference between the minimal and maximal light signal in response to a stimulus, divided by the minimal light signal.
4
Quantification of Nitric Oxide in Colon Tissue
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Supernatants of colon tissue cultures were collected, and nitric oxide (NO) levels were quantified using the Griess Reagent System according to manufacturer’s instructions (Promega, Madison, WI, United States). Briefly, 50 μl of supernatant or nitrite standards were added to 96-well plate, followed by 50 μl sulfanilamide solution and 50 μl N-1-naphthyl ethylenediamine dihydrochloride (NED) solution, with a 10 min incubation period following the addition of each. Absorbance at 550 nm was measured using a Clariostar Monochromator Microplate Reader (BMG Labtech, Germany), and nitrite concentrations were calculated by comparison to the standard curve.
5
Neutrophil ROS Production Assay
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Purified blood neutrophils or sputum cells were resuspended in RPMI medium without phenol red (Gibco, Waltham, MA, USA) at a concentration of 1.5 × 106 cells/ml or 3 × 106 cells/ml, respectively. Prior to stimulation, the cells were incubated for 10 min at 37 °C in the presence of 50 ng/ml tumor necrosis factor-α (TNF-α; Peprotech, Rocky Hill, NJ, USA). Subsequently, the cells were added to a white clear-bottom 96-well plate (Perkin-Elmer, Waltham, MA, USA) and supplemented with luminol (5 mM; Sigma-Aldrich) and one of the following compounds: lipopolysaccharide (LPS) from Pseudomonas aeruginosa (10 µg/ml; Sigma-Aldrich); peptidoglycan (PGN) from Staphylococcus aureus (10 µg/ml; Sigma-Aldrich); N-formyl-methionyl-leucyl-phenylalanine (fMLF) (10–8 M; Sigma-Aldrich). Phorbol 12-myristate 13-acetate (PMA; 150 ng/ml; Sigma-Aldrich) was used as a positive control. An additional condition without luminol was included to determine the background luminescence. The light signal was measured every minute for a period of 3 h by a Clariostar Monochromator microplate reader (BMG Labtech, Orthenberg, Germany). Analysis was performed by subtracting the background luminescence and determining the maximal luminescence as a measure for maximal ROS production. The maximal ROS production of cells stimulated by one of the compounds was normalized to the ROS production of unstimulated cells.
6
Measuring Bacterial Internalization and Clearance
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Cells were incubated with 0.5 mg/ml Texas red‐labeled dextran (10 kD; Molecular Probes) for 60 min at 37°C or on ice in Hank's balanced salt solution supplemented with 20 mM Hepes, pH 7.4, washed with ice‐cold PBS, and lysed in RIPA buffer. Cleared lysates were measured for fluorescence intensity using a CLARIOstar monochromator microplate reader (BMG LABTECH). GFP‐expressing E. coli (E. coli‐GFP) was generated and grown as described.41 Cells were infected with a MOI of 10 in RPMI 1640 (Gibco) full medium (no antibiotics). Cells were incubated at 37°C, washed with ice‐cold PBS and scraped in PBS/5 mM EDTA. To control for bacteria binding to the cells, cells were preincubated in parallel for 30 min with 5 µM cytochalasin D (Sigma‐Aldrich). Cells were analyzed by flow cytometry using a BD LSRFortessaTM cell analyzer (BD Biosciences). Data were analyzed by FlowJo software (Tree Star). To measure clearance of bacteria, THP‐1 cells were incubated with E. coli‐GFP (MOI 10) for 1 h and chased in full medium supplemented with 100 U/ml streptomycin and 50 μg/ml gentamycin. Determination of viable phagocytosed E. coli was done by either fluorescence‐activated cell sorter (FACS) analysis or by lysing macrophages with 1% Triton‐X 100 in PBS for 2 min at room temperature, subsequent incubation on 25 μg/ml chloramphenicol containing agar/LB plates and counting of colony forming units.
7
Quantifying Nitric Oxide in Macrophages
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NO levels from media collected from RAW264.7 macrophages were quantified by Griess Reagent System according to manufacturer’s instructions (Promega, Madison, WI, United States). Briefly, 50 μl of supernatant or standards were added to a 96-well plate, followed by 50 μl sulfanilamide solution and 50 μl N-1-napthylethylenediamine dihydrochloride (NED) solution, with a 10 min incubation period after the addition of each. Absorbance at 550 nm was measured using a Clariostar Monochromator Microplate Reader (BMG Labtech, Germany).
8
MTT Assay for Cell Viability
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Cells were washed and resuspended in culture medium at a concentration of 2 × 105 cells/mL. Then, they were dispensed into 96‐well culture microplates (Falcon®) containing 10 μL/well of serial drug dilutions of ACF. PBS alone was used as control. After treatment at desired times, 10 μL of a 5 mg/mL solution of MTT was added to each well. Plates were then incubated for another 4 hours before the addition of 10% sodium dodecyl sulphate (SDS) solution with 0.01 M hydrochloric acid to solubilize the formazan crystals. After an overnight incubation at 37°C, the absorbance was measured at λ = 590 nm using a spectrophotometer (CLARIOstar® Monochromator Microplate Reader; BMG Labtech, Offenburg, Germany). Living cells were also counted using the trypan blue dye exclusion method.
9
Aqueous Humor PGE2 Quantification
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All aqueous humor samples from both groups were assessed by the same investigator with the same standard preparation to avoid interuser or plate variability. PGE2 concentrations were determined using a commercially available PGE2 Parameter Assay Kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Measurements were performed using a microplate reader (Clariostar Monochromator Microplate Reader, BMG LABTECH, Ortenberg, Germany).
10
Quantifying ATP Levels in Treated Cells
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The cell titer Glo™ (Promega, Madison, WI, USA) reagents were used to measure total ATP levels. Cells were plated into 12-well plates and treated with 6c at 20 μM. After treatment for 6 h, cells were collected and counted. Cells were mixed with cell titer Glo reagent (lysis buffer mixed with cell titer Glo substrate, diluted with PBS 1:1), and the mixture was transferred to 96-well black-walled plates. Luminescence data were recorded using a Clariostar monochromator microplate reader (BMG Labtech, Ortenberg, Germany).
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