Assay principle

The HIF-1a HTRF assay is a sandwich immunoassay involving two specific anti-human anti-HIF-1a antibodies, respectively labelled with Europium Cryptate (donor) and d2 (acceptor). The intensity of the signal is proportional to the concentration of anti-HIF-1a present in the sample. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step. After cell lysis, HIF-1a can be detected using the HTRF HIF-1a cellular assay kit reagents and most TR-FRET multimode plate readers.

Assay Protocol

Two-plate assay protocol Cells are plated, stimulated, and lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of HIF-1a by HTRF reagents. This protocol enables the cells' viability and confluence to be monitored. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step to further streamline the assay procedure. The assay detection can be run in 96- to 384-well plates by simply resizing each addition volume proportionally.

HTRF assay compared to Western Blot

Human HeLa cervical cancer cells were seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2 until 80% confluence was reached. After treatment (6 hours, 10 µM MG-132, 1 mM DMOG), the cells were lysed with 3 mL of supplemented lysis buffer#4 for 30 min at RT under gentle shaking. Soluble supernatants were collected after a 10 minute centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF HIF-1a detection reagents. Equal amounts of lysates were used for a side-by-side comparison of Western Blot and HTRF®. Using HTRF®, just 3120 cells were sufficient for minimal signal detection while 25,000 cells were needed for a Western Blot signal. The HTRF® assay is 8-fold more sensitive than the Western Blot.

HIF-1a and Phospho-Erk1/2 correlation

Human A431 epidermoid carcinoma cells were plated at 100,000 cells/well in a 96 well plate. After a preincubation with 1mM DFO for 30 minutes to prevent the degradation of HIF-1a, the cells were treated for 6 h at 37°C with increasing concentrations of EGF to activate the ERK pathway and the translation of HIF-1a. The medium was then removed and the cells were lysed with 50 µL of supplemented lysis buffer#4 for 30min at RT under gentle shaking. 16 µL of lysate were then transferred into 2 different wells of a 384-sv plate for measurement in parallel.

HIF-1A upregulation upon IGF-1 stimulation

Human MCF-7 epidermoid carcinoma cells were plated at 50,000 cells/well in a 96 well plate. After a preincubation with 1mM DFO for 30 minutes to prevent the degradation of HIF-1a, the cells were treated for 6 h at 37°C with increasing concentrations of IG-1F to activate the AKT pathway and the translation of HIF-1a. The medium was then removed and the cells were lysed with 50 µL of supplemented lysis buffer#4 for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate for detection.

HIF-1A and Phospho-AKT downregulation upon PI3K inhibitor treatment

Human MCF-7 epidermoid carcinoma cells were plated at 50,000 cells/well in a 96 well plate. After a preincubation with increasing concentrations of 1mM DFO for 30 minutes to prevent the degradation of HIF-1α , the cells were treated for 6 h at 37°C with increasing concentrations of the PI3K inhibitor LY294002 for 30 minutes (in triplicate) to inhibit the PI3K/AKT pathway. The cells were then activated with 10 nM IGF-1 and 1mM DFO. Next, the medium was removed and the cells were lysed with 50 µL of supplemented lysis buffer#4 for 30 min at RT under gentle shaking. 16 µL of lysate were then transferred into 2 different wells of a 384-sv plate for measurement in parallel.

HIF1 consists of a constitutively expressed HIF-1ß subunit (87 kDa) and an O2-regulated HIF-1a subunit (120 kDa) which contains an O2-dependent degradation domain (ODD). Under normoxia, HIF-1a is hydroxylated on its ODD domain by O2-activated prolyl hydroxylases (PHDs), leading to its rapid ubiquitination and subsequent degradation by the proteasome.

​Under hypoxia, PHD activity is inhibited and HIF-1a accumulates in the cytosol, translocates to the nucleus, heterodimerizes with HIF-1ß, and is activated. The complex activates the transcription of more than 100 target genes that facilitate responses to the hypoxic environment by encoding proteins involved in angiogenesis, erythropoiesis, cell proliferation/survival, and inflammation.