All Amytracker fluorescent tracers cross the cell membrane of living cells without permeabilization. Of all Amytracker molecules, Amytracker 540 has the best uptake properties. We recommend washing, but if you have sensitive cells, you might consider to skip the washing step. Due to their low background fluorescence and minimal interference with biological autofluorescence, we recommend Amytracker 630 or Amytracker 680 together with Amytracker 540 for live-cell imaging. As Amytracker do not bleach easily, you can use them for confocal, time-lapse imaging or multiphoton microscopy. Use cell culture medium with or without serum according to the requirements of your cell culture.
Solutions and Reagents:
Use Amytracker ex vivo variant, which is provided as 500-fold concentrated solution. The following reagents are required (not supplied):
- Cell culture medium
Assay Procedure:
- Dilute Amytracker in cell culture medium 1:500.
- Incubate your cells in Amytracker supplemented cell culture medium for 30 min under normal culture conditions.
- Replace Amytracker supplemented medium with fresh cell culture medium.
- Perform microscopy method of choice.
Fluorescence Microscopy:
- Amytracker 480: An excitation range of 405-458 nm and a detection range of 470–550 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 480, we recommend using the standard DAPI filter set.
- Amytracker 520: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 520, we recommend using the standard FITC or GFP filter set.
- Amytracker 540: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 540, we recommend using the standard FITC, GFP or YFP filter set.
- Amytracker 630: An excitation range of 458-514 nm and a detection range of 600-650 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 630, we recommend using the standard PI, Cy3, TxRed, mCherry or Cy3.5 filter set.
- Amytracker 680: An excitation range of 530-565 nm and a detection range of 600-800 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 680, we recommend using the standard PI or Cy3.5 filter set.