Application of HEPES in Cell Crawling and Transwell Experiments

Cell crawling and Transwell experiments have a common problem: they are both "multi-step, long-period" processes, and cells and samples need to be repeatedly processed in a non incubator environment. The stability of pH in such experiments often has a greater impact on the results than many people anticipate.

1.Common pain points of two types of experiments

The process of cell slide mounting usually includes: seeding on a cover glass or slide → culturing to an appropriate state → washing → fixation → permeabilization → staining → sealing the slide. The washing, fixation, and staining steps are all completed on a clean bench or at room temperature, and the samples are repeatedly removed from the CO ₂ environment.

Transwell experiments are used for migration, invasion, and co culture studies: cells are seeded in the upper chamber, and the culture medium provides chemotactic signals to the lower chamber. After a certain period of time, the small chamber is removed, non migrating cells in the upper chamber are erased, fixed for staining, and counted. The incubation period of the entire process is long, and there are also multiple sampling and processing steps in the middle.

The problem with both is the same: every time you leave the incubator, the bicarbonate buffer system briefly fails. The impact on short steps is limited, but when accumulated, it may cause the cell state to deviate from expectations.

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2.The position of HEPES in such processes

The buffering capacity of HEPES does not depend on gas-phase CO ₂, with a pKa of approximately 7.5 (25 ℃) and an effective buffering range of 6.8-8.2, which coincides with the growth range of mammalian cells. The general addition amount given in the manufacturer's technical data is 10-25 mM.

In the crawling and Transwell processes, it is mainly used in two places:

Culture medium and operating fluid: Supplement HEPES in the culture system to keep the cells in a stable pH environment when processed outside the box

Washing and incubation solutions: Replace or supplement conventional PBS washing solutions with HEPES containing buffer solutions to reduce pH fluctuations during washing, staining, and other steps

It should be noted that this type of application follows the same principle as cell culture: HEPES is a supplement, not a replacement. The original sodium bicarbonate system in the basic culture medium should be retained, and the two should be used in combination.

3.Three operational suggestions for crawling experiments

3.1 Shorten the single exposure outside the box. Preparing the reagents for washing, fixation, and permeabilization steps in advance, pre warming them in place, and completing them continuously is more effective than simply increasing the buffer concentration. This is the most easily overlooked and easily improved one.

3.2 Pay attention to the pH of the washing solution before fixation. If a liquid without buffering capacity is used for washing before fixation, or if the liquid is left in the air for too long, the pH will move up with the escape of CO ₂. Using a buffer washing solution containing HEPES can make this step more controllable.

3.3 Preheating reagents. Low temperature reagents will bring additional temperature shock, and when combined with pH changes, they will cause greater disturbance to cells. The conventional practice is to balance the detergent and fixative to the operating temperature in advance, but fixative itself has specific requirements and should follow the specific method instructions.

4.Four operational suggestions for Transwell experiment

4.1  Keep the liquid levels in the upper and lower chambers consistent. This is the basic requirement for Transwell experiments. The liquid level difference will form additional hydrostatic pressure, which will affect the migration results; It is also not conducive to the balance of CO ₂ and pH between the upper and lower chambers. It is recommended to visually check after adding the liquid.

4.2  Pay attention to buffer capacity during the long incubation period. The incubation time for migration/invasion experiments is usually several hours to tens of hours. If multiple mid-term observations are required during this period, supplementing HEPES can reduce pH fluctuations.

4.3  The sampling and staining steps are processed centrally. Similar to crawling, the steps of removing, erasing, fixing, staining, etc. from the small chamber are completed in a centralized and continuous manner to reduce intermediate parking.

4.4  Review the osmotic pressure. The addition of HEPES will increase osmotic pressure, and most mammalian cells can tolerate 260-350 mOsm/kg. The migration experiment is sensitive to the osmotic pressure gradient, and after adjusting the formula, NaCl should be measured and adjusted accordingly.

5.Two common considerations

Avoid light throughout the entire process. HEPES can generate reactive oxygen species such as hydrogen peroxide under visible light irradiation, especially in the presence of riboflavin in the culture medium. The staining and observation steps are often accompanied by light exposure, and liquids containing HEPES should be stored away from light

PH is calibrated at the operating temperature. The pKa temperature coefficient of HEPES is approximately -0.014/℃. It is recommended to calibrate the pH meter under conditions close to the actual operating temperature

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The variables involved in the crawling and Transwell experiments are already numerous, with pH being the most easily overlooked and easily improved one. There are only two core approaches: allowing cells to still have buffering capacity when outside the box, and compressing the steps outside the box to the shortest possible length. By implementing these two measures effectively, there is usually a visible improvement in the consistency of the results.

Hubei Xindesheng Material Technology Co., Ltd. has long been dedicated to the research and production of biological buffering agents and related fine chemicals. HEPES and other products can provide supply and customized indicators from gram to ton levels. The company's headquarters is located in Guanggu United Science and Technology City, Gedian Development Zone, Ezhou, Hubei Province. It has two R&D and production bases in Gedian and Huanggang (70 acres), with an annual production capacity of 5000 tons for all categories. Its products are used in IVD in vitro diagnostics, biomedicine, and daily chemical industries.