Serum VEGF concentration was measured by the sandwich enzyme-linked immunosorbent assay kit for human VEGF (Quantikine human VEGF; R&D System, Minneapolis, MN, USA). The assay was performed according to the manufacturer’s instructions. The reaction mixture was quantified using the Bio-Plex protein array reader. The VEGF levels were automatically calculated by Bio-Plex Manager software using the appropriate standard curve. The detection limit of VEGF was 31.2 pg/mL.
Protein array reader
Manufactured by Bio-Rad
The Protein Array Reader is a laboratory instrument designed to detect and quantify proteins in biological samples. It utilizes optical detection methods to analyze protein arrays, which are solid-state platforms containing immobilized protein targets. The core function of the Protein Array Reader is to accurately measure the levels of specific proteins within the sample, providing researchers with valuable data for various applications, such as biomarker discovery, drug development, and disease diagnostics.
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4 protocols using protein array reader
1
Quantitative VEGF Immunoassay Protocol
2
Cytokine and Chemokine Profiling in Lung Tissues
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The levels of cytokine and chemokine expression in the lung tissues were determined by a multiplex cytokine bead array using the MILLIPLEX Rat Cytokine/Chemokine 8-plex assay kit (Millipore, St. Charles, USA), according to the manufacturers' instructions. The reaction mixture was read using the Bio-Plex protein array reader, and the data were analyzed with the Bio-Plex Manager software program.
3
Cytokine Profiling in HIV-Tg and WT Mice
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Plasma samples were collected from HIV-Tg and WT animals at 24 h post-LPS administration and kept frozen until use. Bio-Plex Pro Mouse group 1 Th1 panel L60-00004C6 kit was obtained from Bio-Rad, Portland, ME, USA. Samples from three mice were used for each group. The assay was performed using the Bio-Plex suspension array system according to the manufacturer’s instructions (Bio-Rad). In brief, the appropriate cytokine standards and samples were added to a 96-well flat plate. The samples were incubated at room temperature for 30 min with antibodies chemically attached to fluorescent-labeled magnetic micro beads. After three washes on Bio-Plex ProII wash station, premixed detection antibodies were added to each well and incubated for 30 min. Following three washes, premixed streptavidin-phycoerythrin was added to each well and incubated for 10 min followed by three more washes. Then beads were re-suspended with 125 µL of assay buffer and the reaction mixture was quantified using the Bio-Plex protein array reader. Data were automatically processed and analyzed by Bio-Plex Manager Software 6.0 using the standard curve produced from recombinant cytokine standard.
4
Multiplex Cytokine Quantification in Mouse Serum
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A Bio-Plex Pro test kit was used to measure the mice’s serum concentrations of a number of cytokines (Bio-Rad Laboratories). The assay plate received a total of 50 μl of antibody-conjugated beads. 50 μl of diluted samples, the blank, standards, and the controls were applied to the plate after the samples had been diluted by 1:4. The plate was shaken vigorously at 300 rpm for 30 minutes while being incubated at room temperature (RT) in the dark. The plate received a total of 25 μl biotinylated antibody after three washes with 100 μl wash buffer. The plate was shaken vigorously at 300 rpm for 30 minutes in the dark at RT with 100 μl washing buffer 3 times. The plate was then filled with a total of 50 μl streptavidin-phycoerythrin (PE) and incubated for 10 minutes in the dark at RT with 300 rpm shaking. A Bio-Plex protein array reader evaluated the samples after three washes. Data collection and assay analysis were done using Bio-Plex Manager 6.0 software.
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