Peroxi_SPY555

Labeling Information

1. Prepare 1000x stock solution. Add 50 μL of anhydrous DMSO to the Peroxi_SPY555 vial to prepare the 1000x stock solution (DMSO stock concentration is 1 mM). We recommend using newly or freshly opened and anhydrous DMSO to prepare the 1000x stock solution. In contact with air and moisture, DMSO produces decay products which can strongly reduce the shelf life of the probe in solution, even at -20°C. At this stage, the solution can be colored or not, this has no influence on the performance of the probe. After use, this solution should be stored at -20°C or below. Do not divide the 1000x stock solution into small aliquots, they will decay faster and the probe is not altered by multiple freeze-thaw cycles. When stored properly, this stock solution is stable for 3 months.

2. Prepare the staining solution. Dilute Peroxi_SPY555 to 1x (i.e. a final concentration 1 uM) in your usual cell culture medium (e.g. DMEM + 10% fetal bovine serum) and vortex briefly. If the dilution is not performed in a single step, please use DMSO to prepare the intermediate dilution as using aqueous buffers to prepare the intermediate dilution will lead to the formation of probe aggregates. Proceed quickly to step 3. Since staining efficiency can depend on the cell line, it is recommended to stain cells with 1000x dilution at the first attempt and then optimize the Peroxi_SPY555 dilution factor in further experiments until an optimal staining is achieved. Use only freshly made staining solution, and do not use it multiple times.

3. Cell preparation and staining. Grow cells on coverslips, glass bottom dish or glass bottom multi-well plates as usual. When cells have reached the desired density, replace the culture medium by the staining solution freshly prepared under step 2 ensuring that all the cells are covered with the solution. Place the cells in the incubator at 37°C in a humidified atmosphere containing 5% CO2 for 15 minutes*.

4. Cell imaging. Imaging of Peroxi_SPY555 is best performed using standard Cy3 settings. After labelling, the live cells can be immediately imaged without the need for washing steps. Optionally, a simple washing step consisting of replacing once the labelling solution by fresh culture medium which does not contain the probe may improve the signal to noise ratio but the signal will gradually fade within 30 minutes. If time lapse imaging is performed, it is recommended to keep the probe in the imaging medium during the whole experiment to get a constant signal. 1-2 hours after addition of the probe to the cells, peroxisome metabolization of the probe will lead to increased intracellular background signal and a reduced signal to noise ratio. Therefore, the probe should not be used for timelapse experiments exceeding 2h.

Important Notes:

a) This protocol was optimized and validated using HeLa, U2OS, or HEK293T cells adhering to coated glass or polymer dishes. For other cell lines, recommendations in this protocol should be used as a starting point, and optimal labeling conditions for each cell type should be determined empirically.

b) Peroxi_SPY555 is only mildly fluorogenic and should be imaged by microscopes that are capable of out of focus light exclusion, such as confocal, spinning disk, STED or light sheet microscopes. Peroxi_SPY555 will yield poor signal to noise ratio using widefield microscopy.

* The recommended labelling time weas determined for 2D cultured cells and may differ slightly depending on the cell line used or the sample type (e.g. spheroids, organoids or tissue).