Description
Progranulin, also called proepithelin or prostate cancer (PC) cell-derived growth factor, is the precursor protein of granulin. Progranulin is involved in Frontotemporal Lobar Degeneration (FTLD) and Alzheimer's Disease, where it may protect against beta amyloid toxicity. This cell-based HTRF assay ensures quick results, and is adapted to the functional screening of compounds using progranulin secreting cells.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
Human Progranulin is measured using a sandwich immunoassay involving two specific anti-human Progranulin antibodies, respectively labelled with Terbium Cryptate (donor) and d2 (acceptor). The intensity of the signal is proportional to the concentration of the Progranulin present in the sample.
Assay Protocol
The simple Progranulin assay protocol, using a 384-well small volume white plate (20 µL final), is described on the right. Cell supernatant, sample, or standard is dispensed directly into the assay plate for the detection of human Progranulin. The antibodies labelled with the HTRF donor and acceptor may 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.
Progranulin secretion from iPSC-microglia
iPSC-Microglia were derived as in McQuade et al. 2018. iPS-Microglia were stimulated with 10 ng / mL cytokines for 24 hours in the absence of TGF-b1. Cell supernatants were collected, and secreted levels of Progranulin were measured following the procedure given in the HTRF kit package insert. In agreement with the literature, IFN-γ + IL-1b (pro-inflammatory) treated microglial cells displayed a decreasing progranulin release trend, whereas anti-inflammatory IL-4 and IL-13 Th2 cytokines displayed a significant increase in secreted progranulin.
Data kindly provided by Amanda McQuade and Dr. Mathew Blurton-Jones (University of California, Irvine)
