Description
Perforin (also called Cytolysin or Lymphocyte pore-forming protein) is found in cytotoxic lymphocytes, NK cells (natural killer), and cytotoxic T cells. Perforin creates a pore in the cell membrane to allow Granzyme B to enter the target cells cytoplasm and triggers apoptosis through caspase activation. This kit is designed for the rapid detection of human Perforin in cell supernatant and whole cells. The assay enables high throughput without any washing steps, thereby saving time compared to ELISA.
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 Perforin is measured with a sandwich immunoassay involving two specific anti-human Perforin antibodies, respectively labelled with Europium Cryptate (donor) and d2 (acceptor). The intensity of the signal is proportional to the concentration of Perforin present in the sample.
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, sample, or standard are dispensed directly into the detection plate for detection by HTRF reagents. The antibodies labelled with 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.
Detection of perforin on cell supernatant (two-plate-protocol)
Different cell densities of TALL-104 cells (Effector cells) were plated in suspension under 50 µL in 96-well plate. TALL-104 cells were cultured for 24 hours at 37 °C - 5% CO2. After culture, 16 µL of cell supernatants cells were transferred into a 384-well sv white microplate and 4 µL of the HTRF Perforin and Granzyme B detection reagentswere added. The HTRF signal was recorded after an overnight incubation at room temperature.
