TDP-43 aggregation assay principle

TDP-43 aggregates are measured using a comparison between two HTRF specific signals: the control condition, where soluble TDP-43 only is detected, and the disaggregated condition, where total disaggregated and soluble TDP-43 are assessed. The increase in the disaggregated HTRF signal reflects the level of TDP-43 aggregation.

TDP-43 disaggregation step

Samples containing TDP-43 aggregates require a disaggregation step in order to calculate the level of aggregation of TDP-43 between 2 conditions prior and after sample treatment (sample disaggregation can follow the protocol described on the right). The reagents needed for this step are provided in the kit for maximum convenience. Samples are dispensed directly into the detection (white, low-volume) plate for detection by HTRF® reagents.

TDP-43 aggregation detection

The same control condition sample treated or after the disaggregation step is dispensed directly into the assay plate for detection by HTRF® reagents (384-well low-volume white plate or Revvity low-volume 96-well plate in 20 µl). The antibodies labeled with the HTRF donor and acceptor are pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run up to a 1536 well-format by simply resizing each addition volume proportionally.

TDP-43 aggregation assay data analysis

The aggregated ratio of each sample reflects the presence of TDP-43 aggregates in a sample.

In the case of absence of insoluble aggregates, the aggregated ratio is around 1 or below.

Detection of TDP-43 aggregates in overexpressed TDP-43 WT and mutated HeLa cell model

Human HeLa cells (100,000 cells/well) were transfected in a 96 well plate with 200 ng/well of GFP-TDP-43 Wild Type (full length) or GFP-TDP-43 mutated (C Terminal fragment 220-414 aa) using lipofectamine 2000.

After 48h incubation at 37 °C, 5% CO2, cells were stimulated for 6h with 1 µM of staurosporine (a well established caspase activator cleaving TDP-43 to induce aggregation of C-terminal fragments), or not stimulated.

Then the cell culture medium was removed, and cells were lysed with 50 µL of supplemented lysis buffer #1 (1X) for 30 min at RT under gentle shaking.

Three wells of the same condition were pooled and 2x60 µL of diluted lysate (at ¼ in supplemented lysis buffer) were transferred into a 96 well plate prior to the disaggregation or control detection step, as follows:

• 10 µL of Disaggregation buffer A or Control detection buffer C added
• Incubation 15 min at RT
• 10 µL of Disaggregation buffer B or Control detection buffer C added

After treatment, 16 µL of each sample (disaggregated or control detection condition) were then transferred into a 384 well low volume white microplate before the addition of 4 µL of the HTRF TDP-43 detection reagents. The HTRF signal was recorded after an overnight incubation.

As expected, the overexpression of WT or mutated TDP-43 alone increase the aggregated ratio compared to non-transfected cells. The staurosporine treatment triggers an increase in the TDP-43 aggregation ratio, and this effect is stronger on the mutated TDP-43 overexpressed condition.

HTRF TDP-43 aggregation assay compared to Western Blot

Human HeLa cells were grown for 3 days until 80% confluency was reached, in a T175 flask in complete culture medium at 37 °C, 5% CO2. The cells were stimulated with Staurosporine (1 µM) for 6h before lysis with supplemented lysis buffer.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer.

Each dilution was treated in a disaggregation or control condition, as follows:

60 µL of sample,

10 µL of Disaggregation buffer A or Control detection buffer C added,

Incubation 15 min at RT,

10 µL of Disaggregation buffer B or Control detection buffer C added.

16 µL of each dilution (disaggregated or control detection condition) were then transferred into a low volume white microplate before the addition of 4 µL of the HTRF TDP-43 detection reagents. The HTRF signal was recorded after an overnight incubation. Equal amounts of lysates (not disaggregated) were used for a side by side comparison with Western Blot.

As expected, Western Blot shows C terminal fragments (35 kDa) induced by staurosporine treatment which became insoluble and formed aggregates (data not shown). Therefore the HTRF TDP-43 aggregation assay is 2-fold more sensitive than the Western Blot, at least under these experimental conditions.