All ELISA measurements were obtained using Human BDNF Elisa kit (RayBiotech, Peachtree Conters, GA). For ELISA of cell lines, cells were harvested as described above for western blot and cell lysates were subjected to ELISA per manufacturer’s protocol. For tumor cell lysates and superfusates, animals were injected with HSC2 or NOK cells and tongue tissue harvested at day 9 or day 14 post-cell inoculation. Tissues was weighed, cut, and placed in 500ul of serum free/colorless media for 2 hours at 37C. Superfusates and tissue were separated and stored at −80C until use. Total Protein in superfusates was determined using BCA kit and BDNF release in superfusate samples was quantified following company protocols for the ELISA kit. For tumor tissue lysates, the tissues were subjected to cell lysate preparation as described above for western blot and subsequently subjected to ELISA following company protocols.
Human bdnf elisa kit
Manufactured by RayBiotech
Sourced in United Kingdom
The Human BDNF ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human brain-derived neurotrophic factor (BDNF) levels in biological samples.
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4 protocols using human bdnf elisa kit
1
BDNF Quantification in Cell and Tumor Samples
2
Plasma BDNF Quantification by ELISA
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The remaining whole blood was centrifuged at 2000 rpm 10 minutes and the plasma obtained was frozen at −80 °C until use. BDNF was measured in duplicates using RayBio® Human BDNF ELISA Kit following supplier instructions.
3
BDNF Quantification in SH-SY5Y Cells
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The protein levels of BDNF were determined using a Human BDNF ELISA Kit (RayBio, England) according to the manufacturer's instructions. After a 48-hour exposure to PFOS (10, 50, and 100 μM) or control medium (0.1% DMSO), the supernatants of SH-SY5Y cells cultured in 6-well plates were collected and centrifuged at 1000 rpm at 4°C to remove floating cells, and then the prepared supernatants were immediately added to a 96-well plate coated with a primary antibody specific to human BDNF. The wells were washed, and the secondary biotinylated anti-human BDNF antibody was added, followed by the addition of HRP-conjugated streptavidin. TMB substrate solution was added to the wells as the developing agent, and colour developed in proportion to the amount of bound BDNF. Finally, the Stop Solution changed the colour from blue to yellow, and the intensity was measured at 450 nm. A standard curve was run for each assay, and all standards or samples were run in triplicate.
4
BDNF Quantification in Plasma via ELISA
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BDNF was measured in duplicates in plasma samples using RayBio® Human BDNF ELISA Kit following supplier instructions.
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