Hey Wondong,

your suggestions are right. PBS is just a buffered salt solution (and even contains no Ca2+ or Mg2+) and 30 minutes are quite long for cells in this medium (at least at 37 °C). We stain our cells (Microglia, Macrophages, MEFS) with DCF in HBSS. It contains at least sugar and Ca2+/Mg2+. This is enough to keep the cells healthy for the time of incubation.

Our protocol looks at follows:

1) Centrifuge your cells (we seed them in normal culture plates)

2) Wash them once with HBSS (to remove medium)

3) Add your DCF/HBSS staining solution (10 µM works well in our hands)

4) Incubate at 37 °C for at least 15 and for maximal 30 minutes in an incubator

5) Remove the DCF solution and wash one time with HBSS

6) Detach your cells for FACS and continue with any additional FACS staining

I hope that helps you. Please fell free to ask any further questions.

All the best and stay healthy,

Marc

Reactive oxygen species (ROS) can induce program cell death mechanisms including apoptosis, necrosis, pyroptosis and e.t.c, which lead to sell swelling, shrinking, cellular rupture.

You are very welcome :) Keep me informed how it went.

All the best and stay healthy,

Marc

Marc Herb Hello! I ordered HBSS with Ca, Mg and glucose, but during the shipment, I tested using PBS with Ca, Mg, and added glucose according to the concentration of glucose in alpha-MEM. As a result, cell shrinkage was not observed, and the staining was also much better than previously.

Thank you again, have a nice day :)

Hey Wondong,

yeah good job. How you make the cells happy during incubation does not matter :) We use HBSS because it contains already all the stuff, but mixing is also good. We also do not incubate the cells in media with, for example, serum or phenol red, because we measure ROS nearly always in a plate reader and in this setting serum as well as phenol red highly falsifys your fluorescence.

And a little addition to the DCF probe:

2',7'-Dichlordihydrofluorescein-diacetat (H2DCF-DA) is a cell-permeable derivative of fluorescein. After its diffusion into the cytosol, the two acetate groups are cleaved by cellular esterases generating 2',7'-dichlordihydrofluorescein (H2DCF). This molecule can now be oxidized by ROS to generate the highly green fluorescent 2’,7’-dichlorofluorescein (DCF). H2DCF-DA is often referred to as intracellular ROS probe. However, even after cleavage of the acetate groups, it remains diffusible and therefore does not only remain in the cytosol but also reaches cell organelles and diffuse back into the extracellular space. Therefore only total cellular ROS can be detected with this probe (Ushijima et al., 1997, Hempel et al., 1999).

I am happy that it works. If you like, keep me up to date about your ROS results.

All the best and stay healthy,

Marc

I'm really happy with your professional and helpful answers!

Can I ask one more question?

The official protocol(C6827, ThermoFisher) says, "Remove the loading buffer; return the cells to prewarmed growth medium and incubate at the optimal temperature. For derivatives with acetoxymethyl ester (AM) and/or diacetate groups, allow a short recovery time for cellular esterases to hydrolyze the AM or acetate groups and render the dye responsive to oxidation. The optimal recovery time can vary widely, as some cell types normally exhibit low levels of esterase activity.10"

However, the majority of researches seem to do not provide this recovery time without any problem. If the hydrolysis of esterases is theoretically required, why it is not likely essential? I guess maybe esterases are already acting during the staining time in HBSS, and it is enough itself?

Have a nice day !

Yu

Hey Wondong,

you got it completely right. During your incubation time (15-30 mins), DCF diffuses in the cell and the cellular esterases instantly start cleaving it. That means from this point on, DCF is ready to be oxidized by ROS. Of course this also leads to background, because all cells always produce little amounts of ROS.

The redox balance in the cell is like the pH. It is carefully monitored and regulated by the cell. But after a stimulus (either physiological or chemical) you can increase this ROS production (by various sources in various compartments).

But I do not think you need a recovery time. You can put the cells in fresh medium after incubation, add your stimulus and measure after different time points.

Have a nice day,

Marc

In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".

In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).

If you like, please have a look at:

Functions of ROS in Macrophages and Antimicrobial Immunity

February 2021, Antioxidants 10(2):313

DOI: https://doi.org/10.3390/antiox10020313

Marc Herb Hi! congratulation on your new review "Functions of ROS in macrophages and antimicrobial immunity" instructing versatile roles of ROS and anti-microbial immune system. It always has been a big help to refer to your explanations.

And thanks to your previous help, we also published a paper "The mitochondrial-derived peptide MOTS-c promotes homeostasis in aged human placenta-derived mesenchymal stem cells in vitro" ( https://doi.org/10.1016/j.mito.2021.02.010 ) I'm really glad to deliver good news to you, and I will keep following your inspiring researches keep in the future.

Thanks :)