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22 protocols using stat fax 4200

1

Quantifying Serum Biomarkers via ELISA

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An enzyme-linked immunosorbent assay (ELISA) kit for human S100A4 (Human CircuLex S100A4, Cat No: CY-8086) was purchased from CycLex Co., Ltd., Ina, Japan. ELISA kits for human VEGF (Cat No: SVE00), human syndecan-1 (Cat No: DY2780), and human OPN (Cat No: DY1433) were purchased from R&D Systems. The minimum detection limit for the S100A4 and VEGF ELISA kits were 0.282 ng/mL and 9 pg/ml, respectively. The ELISA plate readings were done using a Stat Fax-4200 microplate reader from Awareness Technology, Inc., Palm City, FL.
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2

Biochemical Assays for Antioxidant Levels

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Venous blood samples of 10 mL were obtained from all participants after a 7- to10-h fasting period. Subsequently, the samples underwent processing and serum separation before being stored at −70°C until further analysis. TAC, CAT, and SOD concentrations were determined via colorimetric assays conducted with NavandSalamat kits from Iran. The analysis was performed using an ELISA reader (Stat Fax 4200, Awareness Technology).
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3

Quantifying Ocular Biomarkers in Vitreous

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Enzyme-linked immunosorbent assay (ELISA) kits for human sVAP-1 (Cat No: ab-119564) and human 8-OHdG (Cat No: ab-101245) were purchased from Abcam, Cambridge, UK. The ELISA kit for human HO-1 (Cat No: KCB3776) was purchased from R&D Systems and the ELISA kit for human HMGB1 (Cat No: ST5101) was purchased from IBL International GMBH, Hamburg, Germany. The ELISA plate readings were done using a Stat Fax-4200 microplate reader from Awareness Technology, Inc., Palm City, FL. The levels of human sVAP-1, 8-OHdG, HO-1, and HMGB1 in vitreous fluid samples were determined based on the standards and protocols provided by the manufacturers.
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4

Spleen Cytokine Quantification by ABTS ELISA

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Spleen cytokines were measured with the ABTS ELISA kit (Cat# 900-K00). According to the manufacturer’s instructions, antibodies for IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and unconjugated antibodies were used for cytokine capture, with a few modifications. Briefly, coated plates (96-well plate, MaxiSorp Nunc Cat. NNC#442404) were incubated with 50 µL (2 µg/mL) of different antibodies overnight. After rinsing three times, (Wash buffer, PeproTech, East Windsor, NY, USA), the plates were blocked (Block buffer, PeproTech) and then washed again. Then, 50 µL of spleen protein (10 µg) were added to the plate in duplicates (diluent solution, PeproTech). The samples were maintained at 4 °C for 2 h and washed three times. An enzyme-substrate reaction was developed with ABTS liquid substrate (PeproTech). All solutions were obtained from the ABTS ELISA buffer kit. The plates were read at a wavelength of 405 nm with a wavelength correction set at 650 nm at different time points in a Stat Fax 4200 microplate reader (Awareness Technology, Palm City, FL, USA).
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5

Quantitative Iron Content Assay

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A number of 2 × 106 treated cells were digested in 100 µL of 5 N HCl solution on a water bath maintained at 80 °C for 4 h. Then, cells were centrifuged at 3000 rpm for 3 min to remove the digested cell debris. In a 96-well plate, 100 µL of cell digest was homogenized with 100 µL of 5% potassium hexacyanoferrate (II) trihydrate (ferrocyanide), and after 15 min the absorbance was read at 630 nm using a Stat Fax 4200 microplate reader (Awareness Technology, FL, USA). To quantify iron content, a calibration curve was obtained from IONP solutions ranging from 0 to 200 mM.
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6

Serum IP-10 ELISA Protocol Standardization

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Serum IP-10 levels were measured using an enzyme-linked immunosorbent assay kit, IP-10 (CXCL10) Human ELISA kit, Invitrogen, Carlsbad, CA, according to the manufacturer’s recommendations. Following centrifugation at 3500 rpm for 10 min, the serum samples were separated and stored at −80 °C until analysis. The IP-10 levels were measured before base treatment initiation and after two months. All the serum specimens were tested in duplicate. Briefly, 50 μL of samples were incubated in ELISA wells of microtiter strips coated with a monoclonal antibody specific for human IP-10. Following incubation at room temperature for 3 h and washings, a biotinylated polyclonal secondary antibody to human IP-10 was added. After washing four times, streptavidin-HRP was added at room temperature for 30 min. After four washes, a stabilized chromogen was added and incubated for 30 min at room temperatures in the dark. The reaction was stopped and absorbance at 450 nm was measured within 2 h in an ELISA reader (Stat Fax 4200, Awareness Technology Inc, Palm City, FL, USA).
A standard curve was produced using freshly prepared serial dilutions of the kit’s reference standard from 500 pg/mL to 7.8 pg/mL.
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7

Quantifying 5-Hydroxymethylcytosine in Prostate Pathologies

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The 5hmC content of genomic WBC DNA in patients with different prostate pathologies and control samples was determined by colorimetric ELISA with the Quest 5-hmCTM DNA ELISA Kit (Zymo Research, Irvine, CA, USA; Cat No. D5426). This is the most cited ELISA-based global 5hmC quantification kit in the literature (over 60 citations according to Google Scholar), and provides scientists with a quick, cost-effective, and reliable alternative to chromatographic methods (e.g., LC-MS/MS, HPLC) that are used for this purpose, particularly for serial measurements, which was the case of our study. Assays were conducted in duplicate per each sample, according to the manufacturer’s instructions by loading 100 ng of DNA per well. To limit variation in measurements related to potential batch or procedural discrepancies, we have randomly rerun ELISA analysis for about a quarter of DNA samples. Optical density (OD) was red at 450 nanometers (nm) using a microplate Reader Stat Fax 4200 (Awareness Technology, Palm City, FL, USA). For absolute 5hmC quantification, a standard curve was obtained by plotting the various concentrations of the positive controls against the corresponding ODs.
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8

LHT-8-17 Cytotoxicity Evaluation in HaCaT Cells

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In vitro LHT-8-17 cytotoxicity was assessed by the reduction of epidermal cells’ metabolic activity as previously described [24 (link)]. The human skin epidermal cell line (HaCaT) was purchased from CLS Cell Lines Service (Heidelberg, Germany). Cells at 1 × 104 cells/well density were seeded in 96-well plates and then incubated with different concentrations (0, 15, 30, 45, 60, 75, and 90 µg/mL) of LHT-8-17 dissolved in PBS. After 24 h of exposure, culture media were substituted with 0.5 mg/mL (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-containing media (MTT, Merck, Sigma–Aldrich, Darmstadt, Germany) and incubated for 4 h at 37 °C. To dissolve forming crystals of formazan produced by survived cells, we used dimethyl sulfoxide. Optical transparency was measured and analyzed for each plate at a 530 nm wavelength using a microplate semi-automatic reader i.e., Statfax-4200 (Awareness Technology, Palm City, FL, USA). Cell viability was presented as a percentage of the appropriate control as the median and SD.
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9

Cytokine Profiling in Wound Healing

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We used sandwich enzyme-linked immunosorbent assay (ELISA) and murine TNF-α, IL-1β, and IL-10 ELISA kits (CUSABIO BIOTECH Co. LTD, Wuhan, China) to assess the tissue cytokine concentration on day 5 of the wound reparation. In brief, fresh granulations were consequently frozen (−20 °C) and thawed trice, and then the homogenate was centrifuged (5× g for 5 min) at 2–8 °C. The supernatant was pipetted in a 96-well microplate with monoclonal anti-TNF-α, anti-IL-1β or anti-IL-10 antibodies. After washing out, unbound components were removed, and a conjugate of polyclonal antibodies to TNF-α, IL-1β or IL-10 with biotin was added to each well. Then, avidin—horseradish peroxidase complex was added to each well for the reaction enhansement. After the TMB chromogenic reaction was stopped, optical transparency was measured and analyzed for each plate at 652 nm on a StatFax 4200 semi-automatic reader (Awareness Technology, Palm City, FL, USA).
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10

Homocysteine, Folate, and Vitamin B12 Assay

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Blood sampling was done at the breakfast and after overnight fasting and the samples were stored at -80°C until analyzed. Serum total homocysteine concentrations were determined on frozen samples by high-performance liquid chromatography (HPLC) method (KNAUER, Germany), coupled with fluorescence detector.[14 (link)] For evaluating folic acid and vitamin B12, the samples were collected in tubes protected from light and levels of folate and vitamin B12 were measured simultaneously in the frozen serum aliquot by a double labeled radioassay kit (ICN Pharmaceuticals, New York) (Stat Fax 4200, Awareness Technology, USA) in all cases and controls.
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