HTRF Human Total FLT3 assay principle

The HTRF Human Total FLT3 assay quantifies the expression level of FLT3 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Human Total FLT3 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of FLT3 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Human Total FLT3 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Human Total FLT3 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Human Total FLT3 one-plate assay protocol

Detection of Human FLT3 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Specificity of the Total FLT3 assay

HEK293 cells were transfected with 100 ng of human FLT3 plasmid, empty plasmid, or non-transfected, in a 96-well plate (100,000 cells/well) under 100 µL for a 24h incubation at 37°C, 5% CO2. After medium removal, cells were lysed with 50 µL of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking, and 16 µL of lysates were transferred into a low volume white microplate before the addition of 4 µL of premixed HTRF (h) Total FLT3 detection antibodies. The HTRF signal was recorded after 3h of incubation at RT.

The HTRF (h) Total FLT3 assay efficiently detects FLT3 in the human FLT3 transfected condition compared to the cells transfected with the empty plasmid.

No signal was observed with the non transfected cells or the empty plasmid transfected cells , demonstrating the specificity of the kit.

Validation of HTRF Human Total FLT3 kit on endogenous human leukemia cell lines and HEK293 transiently over-expressing human FLT3

Human leukemia THP-1 cells, human leukemia MV4-11 cells, and human FLT3 transfected HEK293 cells were cultured in T175 flasks in a complete culture medium for 24h at 37°C, 5% CO2. The cells were lyzed with supplemented lysis buffer # 4 for 30 minutes at RT under gentle shaking, at a final density of 25 x 10.6 cells/mL for the THP-1 and MV4-11 cells and at a final density of 3 x 10.6 cells/mL for the transfected HEK293 cells.

The FLT3 protein expression level was assessed with the HTRF (h) Total FLT3 kit. Briefly, 16 µL of cell lysate were transferred into a low volume white microplate followed by 4 µL of premixed HTRF detection reagents. The HTRF signal was recorded after a 3h incubation at RT.

The THP-1 cells expressed endogenous Wild-Type FLT3 (FLT3 WT), the MV4-11 cells expressed endogenous Internal Tandem Duplication (ITD) FLT3 mutant, and the HEK293 cells over-expressed FLT3 WT. FLT3 proteins were detected in all tested human cell lines at different levels.

For a determined cellular model, cell density optimization is mandatory in order to be within the dynamic range of the kit. The HTRF (h) Total FLT3 assay efficiently detects endogenous and over-expressed, WT FLT3, or ITD FLT3 mutant, in various human cellular models expressing different levels of the protein.

HTRF Human Total FLT3 assay compared to Western Blot

Human FLT3 transfected in HEK293 cells was cultured in a T175 flask in a complete culture medium for 24h at 37°C, 5% CO2. The cells were lyzed with 3 mL of supplemented lysis buffer # 4 for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF (h) Total FLT3 detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF (h) Total FLT3 assay, 200 cells/well were enough to detect a significant signal, while 1,500 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF (h) Total FLT3 assay was 7.5 times more sensitive than the Western Blot technique.

FLT3 Signaling Pathway

FLT3 is a class III receptor tyrosine kinase expressed in immune cells and activated by its ligand, FLT3L. After FLT3L binding, the FLT3 receptor dimerizes and activates a variety of pathways, including the phosphatidylinositol 3-kinase (PI3K) and the MAPK pathways resulting in cell division, proliferation, and survival of immune cells. A dysregulation of the FLT3 pathways could lead to anarchic proliferation, immature immune cells, and oncogenic phenotypes. Mutations in the FLT3 gene lead to constitutive activation of the receptor kinase activity or to a disruption the autoinhibitory activity of the receptor. The ITD FLT3 mutant does not require the FLT3L to activate its downstream pathways. FLT3 is well known for its implication in the development of leukemia and more specifically in Acute Myeloid Leukemia (AML).