How to’s

Protocol for using azidoblebbistatin

Question:

Recently, we have done some tests of azidoblebbistatin on hela cells using A1R multiphoton microscopy (Nikon Ti-E). This instrument own 800 nm wavelength with 2700mW power and 900nm with 1900mW. We have test the two parameters with different stimulate time from 1s to 8 min. But we did not observe the stimulate area to turn green. So we want you give us a detailed protocol, which containing the concentration of the drug, stimulate time, the wavelength and its power. 

(Anna Rauscher, Phd answers)

Based on recent experiments the suggested wavelength for 2-photon irradiation is 860 nm. Based on the dimensions of the 2-photon laser focus, suggested step size is 0.1 um/ pixel.

In most cases fast scanning is recommended.

It is necessary to optimize the followings:

  1. intensity of laser and irradiation time
  2. azidoblebbistatin concentration

Below you find an example of a working protocol on HeLa cells for a 2.4 um x 10-15 um area:

5 uM azidoblebbistatin (incubation time: 15 mins)

3.5% laser intensity (~20 mW in the focal spot) 860 nm

166.1um/ms scanning speed

0.1 um/ pixel step size

500 ms scanning of the area, repeat 10 times, every 2 sec

It is important to check if your cells tolerate this laser intensity to make sure that no photodamage occurs. If your cells tolerate more, you may need to increase the intensity. In the above example 5 uM azidoblebbistatin was applied, which fully inhibited myosin in the irradiated area after irradiation. Without irradiation, 20 uM para-nitroblebbistatin had to be used for full inhibition.

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About blebbistatin derivates

Which blebbistatin derivative should I use?

(András Málnási-Csizmadia answers)

I think p-aminoblebb is a useful compound because it is highly soluble in water, non-fluorescent, stable and non-toxic. Especially its high solubility makes it very useful because its pipetting is much more reproducible compared to blebbistatin.

Nevertheless, its less hydrophobicity slows its penetration to the cell. Also, please note that its IC50 is a little bit higher compared to blebbistatin.

In summary, in many cases, especially in the in vivo experiments or when you want a longer (more than 20 min) treatment it is very useful because its concentration remain stable.

If you want fast cell penetration and lower IC50, I recommend p-nitroblebbsitatin which is also non-toxic, stable and non-fluorescent, however, its water solubility is approximatelly the same as that of blebbistatin. However, even in this aspect p-nitrobleb is much better than blebbistatin, because blebbistatin forms tiny crystals during its precipitation which completely covers the microscopic image, while p-nitrobleb produces slowly large crystals, therefore they practically do not disturb the image.

(reference for p-nitrobleb: Angew Chem Int Ed Engl. 2014 Jul 28;53(31):8211-5. Képiró M1, Várkuti BH, Végner L, Vörös G, Hegyi G, Varga M, Málnási-Csizmadia A. para-Nitroblebbistatin, the non-cytotoxic and photostable myosin II inhibitor. )

So, depending on your experiment you may use p-nitrobleb or p-aminobleb. In the in vitro cellular tests most of the time I use both in order to exclude possible artifacts.