HTRF BDNF Detection Kit, 500 Assay Points

BDNF assay principle

BDNF is measured using a sandwich immunoassay involving two specific anti-BDNF antibodies, respectively labeled with an HTRF donor and acceptor dyes. The intensity of the signal is directly proportional to the concentration of BDNF present in the sample.

BDNF assay protocol

The assay protocol, using a 384-well small volume white plate or a Revvity low volume 96-well plate (20 µL final), is described on the right. 16 µL of cell supernatant or standard are dispensed directly into the detection plate for detection by HTRF® reagents. The antibodies labeled with the HTRF donor and acceptor can be pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run in 96- to 384-well plates by simply resizing each addition volume proportionally.

BDNF standard curve

Recombinant BDNF provided in the kit (standard) was serially diluted in diluent #5, following the procedure given in the kit’s package insert. The HTRF signal, expressed as a delta ratio, was plotted as a function of the BDNF concentrations tested.

Intra-assay (n=24)

Each of the 3 samples was measured 24 times, and the % CV was calculated for each sample.

Intra-assay (n=5)

Each of the 3 samples was measured in 5 different experiments, and the % CV was calculated for each sample.

Human microglial HMC3 cell line treated with IFN-γ

The human microglial cell line HMC3 was seeded in a T75 flask (3 million cells) in complete medium, and cultured until 80% confluence was reached. Cells were then treated or not with 10 ng/mL of human IFN-γ for 24h. Cell supernatants were collected, and secreted levels of BDNF were measured following the procedure given in the HTRF kit’s package insert.

In accordance with the literature, IFN-γ treated microglial cells displayed a decrease in BDNF release.

hiPSC-derived astrocytes Astro.4U treated with pro-inflammatory cytokines

Human iPSC-derived astrocytes (Astro.4U®, Ncardia) were plated in a 96-well poly-L-ornithine/laminin-coated plate and cultured for 7 days. Cells were then treated or not with a cocktail of human pro-inflammatory cytokines (10 ng/mL TNF-α + 100 ng/mL IL-1β + 100 ng/mL IFN-γ) for 24h. Cell supernatants were collected, and secreted levels of BDNF were measured following the procedure given in the HTRF kit’s package insert.

As expected, the concentration of BDNF released by astrocytes increased significantly when cells were exposed to the cocktail of pro-inflammatory cytokines.

Simplified pathway for BDNF

Astrocytes are very sensitive to their environment and respond quickly in response to CNS damage. Activated signaling pathways lead to pro- or anti-inflammatory phenotypes. 

In response to some cytokines and growth factors, astrocytes activate the Jak/STAT3 pathway, which is thought to be responsible for the upregulation and secretion of many neurotrophic and neuroprotective factors promoting neuron survival and synapse repair, including BDNF.