Pitstop® 2, Novel cell-permeable clathrin inhibitor (ab120687)

As stated on page 7 of our protocol booklet for ab120687, longer incubation times (such as greater than 30 minutes) may lead to non-specific effects and are therefore not recommended.

To answer this question I think its important to highlight the following note found on page 4-5 of the protocol booklet:

-The final working concentration of DMSO should generally be between 0.3 and 1%. Low concentrations of DMSO (i.e. 0.1%) may lead the compound to precipitate out of solution. However, please note that the amount of DMSO needed to keep the compound in solution is dependent on the concentration of the compound. For example, in 1% DMSO, drug concentrations of 300 μM or higher may also lead to precipitation (depending on which aqueous buffer is used and the temperature.

-Pitstop 2™and the Pitstop 2™negative control are not water soluble, but can be used in low concentrations of DMSO (up to 1% for cellular experiments, up to 3% for in vitro use, see above and below) after serial dilution from the 100% DMSO stock solution.

To summarize, a minimum of 0.3-1% DMSO is required in the tissue culture medium to maintain solubility of Pitstop2.

Thank you for your inquiry.

I heard back from the lab and our chemists believe that this should have had no effect on the stability of the compound. Please tell the customer that the compound should be fine to use.
We will remove the ‘do not freeze’ message on the datasheet.
I hope this information helps. Please contact us with any other questions.

We recommend use of serum free media (containing 10 mM HEPES; pH 7.4). Up to 0.1% serum can be tolerated for some cell lines, however this has not been extensively investigated. Longer incubation times may lead to non-specific effects and is therefore not recommended. Generally, 5-10 minutes at 37C is enough for effect.

Unfortunately as this product is quite novel we were unable to find any specific citation of this product being used in Ovarian cancer cells. And as such, we were unable to find an ID50 value.

However, we have a protocol book of guidelines for Pitstop 2 which may be of some help for the concentration:

https://www.abcam.com/ps/products/120/ab120687/documents/ab120687%20Pitstop%202%20%28Web%29.pdf

In this booklet, for complete inhibition of clathrin mediated endocytosis we recommend using a final concentration of 20-25 uM.

We cannot find any reason why the customers cells should be dying (we have not received any other reports of the compound being toxic to cells). Previous publications have only looked at HeLa and COS-7 cells ranging in concentrations between 20 - 30 uM (please see attached). As far as we are aware U251 cells are epithelial glioblastoma cells and should be comparatively resilient.
Are you performing a DMSO control and that it is not the DMSO that is cytotoxic? Attached is a paper where they state that the viable DMSO concentration in these cells is 0.1%. As a guide, for a stock solution of 30 mM, you would need to dilute a 5 mg pack into 352 ul of DMSO.

The chemist here thinks that the white puff you are seeing is simply the DMSO meeting the PBS, and suggests that you will see the same puff when you pipet just DMSO, without any Pitstop 2™ dissolved in it. You might try that, as an easy check. He doubts the Pitstop 2™ comes out of solution, and that whatever you are seeing will disperse.

I think heating the PBS would not affect the inhibitory activity but I have no data demonstrating that this is the case. I would observe how the DMSO solution appears when pipetted into heated (eg, 40C) PBS, and compare that appearance to what happens when you pipet into a room temperature PBS preparation. If you see a white puff in both cases, use the RT prep. If you see no white puff with the heated prep, use that one. Ideally, you would compare the inhibition results from both preps but I understand this is not what you were planning on.

Please let me know if you still have concerns, or other questions.

Thank you for contacting us.

Here are some guidelines for reconstituting and storing ab120687:

A stock solution should be prepared by dissolving in 100% DMSO (fresh, sterile) to give a final concentration of 30 mM.
Vortexing should be used to ensure solublization of the compound.
This stock solution is stable at room temperature for about 4-6 hrs.
Preparing initial stock solutions that are too low in concentration limits later use of the compound in aqueous buffers, as the DMSO concentration in the final aqueous solution should not exceed about 0.1%.
Aliquots of the stock solution should be stored at -20°C.
Stock solutions should not be left at room temperature for longer than necessary to prepare the working solutions.
Avoid freeze/thaw cycles of the stock solution as this may affect the activity of the compound.
The compound will remain soluble in 1% DMSO. However, at concentrations of 300 µM or higher, precipitation may occur (depending on which aqueous buffer is used and the temperature).

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for contacting us. The product ab120687 is not available reconstituted in DMSO, I am sorry to report. Here are some guidelines for reconstituting and storing it:

A stock solution should be prepared by dissolving in 100% DMSO (fresh, sterile) to give a final concentration of 30 mM. Vortexing should be used to ensure solublization of the compound. This stock solution is stable at room temperature for about 4-6 hrs. Preparing initial stock solutions that are too low in concentration limits later use of the compound in aqueous buffers, as the DMSO concentration in the final aqueous solution should not exceed about 0.1%. Aliquots of the stock solution should be stored at -20°C. Stock solutions should not be left at room temperature for longer than necessary to prepare the working solutions. Avoid freeze/thaw cycles of the stock solution as this may affect the activity of the compound. The compound will remain soluble in 1% DMSO. However, at concentrations of 300 µM or higher, precipitation may occur (depending on which aqueous buffer is used and the temperature).

Please let me know if these notes do not help.

Thank you for contacting us.

As we do not undertake our own research we are only able to provide information available in the current literature. Von Kleist et al., (2011) found no cytotoxicity in HeLa cells after 8, 20 or 72 hours and concluded that the Pitstop compounds act as selective inhibitors of clathrin TD function and do not adversely affect cell viability.

A great advantage of Pitstop 2TMis that its effects on endocytosis are reversible, however in your situation this is not required. We do not have any information about the effect of PBS washes on Pitstop 2 binding , however the guidelines for protocol use state that incubation of Pitstop 2TM-treatedcells for 45-60 min along with two changes of media in full serum containing medium will restore the ability of cells to undergo CME.

The attached paper by Hill et al., (2010) states that Iminodyn-22 is made up in a serum containing buffer and left for 30 minutes prior to the reaction being stopped by the addition of EDTA. A 30 minute incubation for iminodyn-17 was also used. They also states that cell morphology is unaffected by an exposure of greater than 30 minutes.

I hope that this has been helpful. This information was found in both of the attached papers and also the guidelines for use for Pitstop.



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