Membrane labeling protocol

abberior STAR membrane probes for live-cell imaging

abberior offers a variety of excellent fluorescent dyes with optimized properties for the labeling of biomolecules, spectroscopic studies, optical microscopy, and particularly optical nanoscopy featuring STED super-resolution.

Our abberior STAR membrane probes can be readily used to specifically label the outer plasma membrane of living cells. All our bright and photostable abberior STAR membrane probes are modified by cholesterol as a functional group. Since the dye itself is not incorporated into the membrane, our unique membrane probes do not change the composition of the plasma membrane by foreign substances. The natural behavior of the membrane and fluidity will maintain.

In combination with our abberior LIVE dyes, you get a toolbox of super-efficient probes, that provide specific labeling of intracellular targets and outer cell membrane, making long-term multi-color live-cell imaging possible. Additionally, extremely low nanomolar probe concentrations reduce potentially toxic effects and cellular stress

Storage

Our abberior STAR membrane probes are freeze-dried and shipped at room temperature. Upon arrival, the product can be stored for up to one year at –20 °C. Proximately before the staining procedure dissolve the probe in DMF or DMSO. Once dissolved the stock solutions should be kept at –20 °C, protected from light and moisture.

Note: Depending on solvent quality the shelf-life of the stock solution might be significantly reduced compared to the probe in its solid form – even if stored at –20 °C.

Staining of the outer plasma membrane using abberior STAR membrane probes

The procedure described below has been successfully tested with our abberior STAR membrane probes in several adherent mammalian cultured cell lines. These procedures may not be optimum for certain experimental conditions but have yielded consistent results in most instances.

Required reagents / equipment; not provided

• water-free DMF or DMSO
• glass coverslips, glass-bottom dish, or similar imaging chamber with a glass thickness of _~170 µm (No. 1.5 or No. 1.5H)
  
  Note: We do not recommend using plastic coverslips or live-cell chambers with plastic bottoms because only suboptimal imaging results are achieved. If possible, coverslips with grids, gratings, or similar should be avoided, as these structures can interfere with imaging causing aberrations that degrade image quality
• Live-cell imaging medium (e.g. evrogen DMEMgfp-2)
• Tweezer
• Optional: Cavity slide or coverslip holder
• Optional: silicone glue (e. g. Twinsil, Picodent)
• Fluorescence microscope with live-cell incubator and suited excitation light and detection filter

Staining procedure for cultured cells

Depending on the doubling time of the cell line, the seeding time must be determined. Seed cells in a desired density in a cell culture chamber or onto coverslips before labeling. Use a standard cell culture medium that is optimal for the cell line.

Note: Cells grown in very high densities, i.e., a confluent layer, may give rise to high labeling background.

Staining and imaging will take place on the same day

1. Prewarm the live-cell imaging media to the optimal temperature required to cultivate the desired cell line. In most cases, this would be 37 °C.
2. Prepare a stock solution of 10 µM by dissolving the probe in 500 µl of DMF or DMSO.
   
   Note: If dissolved in DMF or DMSO the substance can precipitate at -20 °C. It will be redissolved after the solvent has been warmed up to room temperature.
   
   Note: If you´re using an evaluation sample, dissolve the substance in 10 µl DMF or DMSO to receive a concentration of 10 µM.
3. Prepare the staining solution using a prewarmed live-cell imaging medium. A final concentration of 10 to 50 nM is recommended. The required concentration for proper labeling strongly depends on the used cell type.
   
   Optional: Our direct LIVE labels can be combined with our membrane probes. Simply add them to the staining solution for multicolor live-cell imaging.
   
   Note: The staining solution is not stable for extended periods of time. Therefore, it is recommended that you only prepare enough solution for immediate use.
4. Remove the cell culture medium and rinse the cells once in the prewarmed live-cell imaging medium.
5. Remove the medium and add enough staining solution to the cells. Incubate for 10 to 30 min at optimal cell growth conditions (temperature, humidity, CO₂-controlled environment).
   
   Optional: Afterward, cells can be rinsed with a fresh live-cell imaging medium. abberior STAR membrane probes are used in very low nanomolar concentration, meaning the background signal is already reduced. Additional washing steps might reduce the background signal further.
6. Cells are embedded in a fresh live-cell imaging medium.
   
   If coverslips were used: simply take the coverslip out of the staining solution using tweezers. Place the coverslip in a
   
   i. coverslip holder or
   ii. mount it onto a cavity slide (cells facing downwards) which is filled with fresh live-cell imaging medium.
   
   In case of (ii) remove the excess imaging medium using tissue paper. Gently press down the coverslip to prevent it from moving. The mounted sample can be sealed using silicone glue (e. g. Twinsil, Picodent).
7. After staining and embedding, the samples should be immediately imaged on a microscope equipped with a live-cell incubator.
   
   Note: For live-cell imaging, cells must always be kept at ambient conditions (temperature, humidity, pH, and CO₂-conditions). This is particularly important for long-term measurements.

Abbreviations

DMF N,N-Dimethylformamid
DMSO Dimethylsulfoxide
STED Stimulated Emission Depletion