Live-cell imaging

Live-cell imaging

All Amytracker variants cross the cell membrane of living cells without permeabilization. Due to their low background fluorescence and minimal interference with biological autofluorescence, we recommend Amytracker 630 or Amytracker 680 for live-cell imaging. As Amytracker do not bleach easily, they are excellently suited for repeated illumination during time-lapse imaging. If possible, use serum-free medium during incubation.

Solutions and Reagents:

  • Amytracker - Aqueous or Amytracker - DMSO
  • Imaging medium: Serum- and Phenol Red free cell culture medium

Assay Procedure:

  • Dilute Amytracker in Imaging medium 1:1000.
  • Incubate your cells in Imaging medium for 30 min under normal culture conditions.
  • Image cells
  • Replace Imaging medium with standard cell culture medium.

Readout:

  • Perform imaging using the filter settings listed in the table below.

Literature summary of live-cell applications

Amytracker molecules are small, non-toxic, and live-cell compatible. They become fluorescent upon binding to protein aggregates, enabling direct visualization and tracking of protein aggregation over time. Their use in live-cell research has been demonstrated in numerous publications, several of which are summarized below.

Table: Published examples of Amytracker use in live-cell research. Information is summarized from the cited publications.
Publication Cell type Amytracker variant Target Staining conditions Detection method
Ariyath 2026 MC65 cells (neuroblastoma) Amytracker 680 Aβ aggregates 30 min incubation at 37°C, in Opti-MEM. Excess dye was removed with three washes. Fluorescence readings were collected daily for eight days. Fluorescence microscopy on day 6. Microplate reader & fluorescence microscopy
Eltom 2024 hiPSC-derived astrocytes Amytracker 680 Tau fibrils Cells were exposed to brain-derived AD fibrils at a concentration of 25 mg starting tissue/ml in culture medium for 3 days. The cells were then washed 3 × 5 min with PBS and subsequently stained with Amytracker 680. Following a 30-minute incubation, the cells were washed 3 × 5 min with PBS and tau free culture medium was added. Time-lapse microscopy, confocal fluorescence microscopy
Raymundo 2024 HaCaT cells Amytracker 630 Amyloid-like aggregates Not specified in the publication. Confocal fluorescence microscopy
Li 2024 Macrophages Amytracker 680 Aβ fibrils The sonicated Aβ fibrils (4 μM total monomer concentration) were first tagged with a 1:1000 dilution of Amytracker 680; this was then delivered to the macrophage surface. Fluorescence microscopy
Pinzi 2024 Primary neurons from mouse dorsal root ganglia (DRG) Amytracker 680 Amyloid fibrils Serum-reduced DMEM containing NGF and without phenol red one day prior to live imaging. Amytracker 680 was diluted 1:500 in the culture medium. After 30 minutes of incubation, cells were imaged with a 453-nm laser. Fluorescence microscopy
Chandhok 2023 MCF-7 cells (Breast cancer cell line) Amytracker 680 Aβ (1–42) aggregates Amytracker dye (red) was added to the media of live cells and images were captured. MCF-7 cells (ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (high glucose) which was supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin–streptomycin, which were incubated at 37 °C and 5% carbon dioxide. Confocal fluorescence microscopy
Juliani 2023 HEK293 cells Amytracker 680 Prion aggregates AmyTracker 680 (Ebba Biotech) labeling of live cells was performed with 1:1000 of the dye in Opti-MEM for 30 min (37°C, 5% CO2) followed by replacing with fresh medium. Fluorescence microscopy
Hochmair 2022 HEK sensor cells Amytracker 680 Tau aggregates Likely live-cell staining, but fixation status not explicitly stated. Fluorescence microscopy
Choi 2022 SNCA-A53T hiPSC-derived neurons Amytracker 540 α-Syn aggregates Cells were washed twice and loaded with 1% Amytracker for 20 min. Room temperature HBSS (Hank’s Balanced Salt Solution) was used as a recording buffer. Confocal fluorescence microscopy
Morten 2022 HEK293 cells Amytracker 630 α-Syn, tau, Aβ aggregates Live-cell imaging of these cells incubated with 1 μM aggregates was conducted in FluoroBrite Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. TIRF microscope
Hofbauer 2021 Macrophages Amytracker 480 Amyloid fibrils Cells were washed with PBS and incubated with the AmyTracker 480 reagent (1:1000, 1 h) at 37°C and 5% CO2. As assessed by flow cytometry, an increase in amyloids was defined as increase in fluorescence. Flow cytometry

Multi-Laser / Multi-Detector Imaging with Amytracker

Amytracker are optotracers with structure-dependent photo-physical properties. All Amytracker variants are designed to bind to the Congo red binding pocket on the amyloid fibril and require a theoretical minimum of eight in-register parallel-β-strands for binding. Therefore, Amytracker reliably labels amyloids derived from a variety of amyloidogenic proteins or peptides from different species. Due to their structure-dependent photo physical properties, the Amytracker variants are only fluorescent when binding to a target and different targets can produce a difference in the molecules fluorescence spectrum. To investigate different targets, we recommend to perform imaging by exciting the sample with different wavelengths collecting fluorescence intensity in multiple emission ranges (see the table below for reference). Excitation- and emission spectra for all Amytracker variants can be accessed here.

Table: Multi-laser / Multi-detector imaging protocol performed on a Zeiss LSM800 CLSM.
Channel Excitation Emission range Amytracker variant
CH1 405 nm 400-490 nm Amytracker 480 Amytracker 680
CH2 405 nm 490-600 nm Amytracker 480 Amytracker 680
CH3 405 nm 600-660 nm Amytracker 480 Amytracker 680
CH4 405 nm 660-700 nm Amytracker 480 Amytracker 680
CH5 488 nm 500-580 nm Amytracker 520 Amytracker 540 Amytracker 630
CH6 488 nm 580-650 nm Amytracker 520 Amytracker 540 Amytracker 630
CH7 488 nm 650-700 nm Amytracker 520 Amytracker 540 Amytracker 630
CH8 561 nm 600-650 nm Amytracker 630 Amytracker 680
CH9 561 nm 650-700 nm Amytracker 630 Amytracker 680
CH10 Brightfield
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