After the 48 hours in the incubator, the pH of the sample without HEPES had increased to 9.8, while the sample with HEPES had increased to 9.4. Gen5™ Microplate Reader and Imager Software, besides controlling reader function, can be used to calculate pH from previously established pH calibration curves. The most common CO2 percentage is 5% which is fine for most cell culture media's but as already stated DMEM should always be used at 10% CO2. Ths led to about 10% CO2, and 3.7 g/L sodium bicarbonate. Medical physiology principles for clinical medicine (4th ed., International ed.). Phenol red powder (cat # P-3532) was from Sigma Aldrich. Have you monitored the pH during the 48 hour period as well? I found powder forms where I can add the desired amount of NaHCO3 but I don't want to spend time on preparation, pH adjustments and sterility issues. If you remove the high CO2, the sodium bicarbonate takes over and you get a basic pH. Figure 4. If you are not currently subscribed, you may sign up now to begin receiving issues as they are released. Figure 9  . Change in pH levels with DMEM after loss of CO2 supply. Alternatively, if you have no CO2 control in your incubator and do not want to add too much HEPES, you may go for other components such as MES, which have lower pKa values and therefore yield lower final pH of your medium. The use of a second wavelength to either correct for plate absorbance or provide a ratiometric determination has the added benefit in that both will correct for condensation or bubbles in the well that may form over time. While concentrations vary with different media formulations, when present it is typically in the 5-15 mg/mL range. At low pH levels, phenol red turns the medium yellow, while at higher pH levels it turns the medium purple. Your DMEM probably has 3.7 g sodium bicarbonate which will provide a pH of 7.4 in 10% CO2. You need CO2 for proper buffering of DMEM. DMEM is commonly used with 5% CO2, but its optimal buffering capacity is at 10%. At high pH the media has a visible fuchsia color that changes to red then yellow as the pH deceases. As the concentration of phenol red solution was equivalent to the reported recipe of the media formulation, the very near match in the shape and … Closer examination of the two absorbance peaks in media with and without phenol red reveals an influence from the media constituents to the absorbance at 415 nm (Figure 5), whereas the more substantial peak at 560 nm seems to not be affected by media components. DMEM Gibco™, haute teneur en glucose, sans glutamine, aucun rouge de phénol n’est utilisé dans les applications de traitement de culture cellulaire. Above 8.2 the dye turns a bright fuchsia color. 3.7g/L of NaHCO3 requires 10% CO2. You can also register to be invited to future webinars here. Figure 2. Change in absorbance after cessation of CO2 supply with different media formulations. I have noticed this bicarbonate and CO2 relation by observing the change in medium colour. No. -Sodium Pyruvate is present in DMEM with phenol red and absent in DMEM without phenol red. The original DMEM formula composition contains 1000 mg/L of glucose and was first reported for culturing embryonic mouse cells. I am using ACHN and A498 (renal cell carcinoma cell line). As shown in Figure 12, tissue culture media rapidly increases in pH by approximately 2 pH units in the absence of carbon dioxide gas over time. The BioSpa system controls reader scheduling and maintains cells in a humidified controlled environment (37 °C, 5% CO2) in between imaging and plate absorbance measurements. Search our Accessory database for Accessories compatible with your instrument. I found one from ATCC (1.5g/L), but they only have high glucose. Absorbance data at 560 nm obeys Beers law in regards to pathlength. Absorbance at 560 nm for different media formulations. This method of correction has the added benefit of correcting for specific well-to-well variances, whereas defined blanks can only accommodate an average plate background. Click here to request a quote on a product, software, peripheral or service contract. Wavelength with absorbance peak in the presence (+) or absence (-) of phenol red is indicated with an arrow. For example 5mM HEPES, 5mM MOPS and 5mM BES will give you 15 mM buffering, without the high concentration of 1 buffer. The extent of pH increase can be determined by interpolating data from calibration curves. without the usual CO2 and a visible change of media colour may cause wrong or questionable measurements. Using a 415/560 ratiometric analysis will provide a greater dynamic change, but results in less accurate pH determination. I can see that the medium color becomes more pinkish, but I don't know how much the pH has changed. HEPES should have helped that out, but it looks like it did not. I have used this and it works well, and it also means that you can leave the cells outside the incubator for a longer as well. The initial pH of the no-HEPES sample was 7.4; the sample with HEPES had a pH of 7. α-MEM (Minimum Essential Medium Eagle - alpha modification) is a medium based on MEM published … What could be the reason and will it effect the growth of my cell line? Alternatively, the ratio of the 415 nm and 560 nm peaks can also be used as a marker for pH change. If you use HEPES in an OPEN environment then the pH will drift. As observed in Figure 3, complete media has a large peak in absorbance in the UV range and two lesser peaks centered on 415 nm and 560 nm. Non-humidified experiments were performed directly in a Cytation 5 Cell Imaging Multi-Mode Reader supplied with 5% CO2 or not as indicated. Lausanne University Hospital - University of Lausanne, Switzerland. Similar results are observed when live cells are present in a microplate that has been subjected to a loss of carbon dioxide gas. As already explained the medium with higher sodium bicarbonate needs a Co2 incubator for 5%Co2. When the 560 nm absorbance at different pH levels for DMEM, McCoy’s 5A and RPMI 1640 were examined, all three had marked increases with reduced pH. Specific supplement concentrations are assay dependent. 0.001), and the acidic and basic forms of the indicator showed similar activity. A very rapid color change would be indicative of some sort of microbial contamination. The BioTek Global Technical Support Center (GTS) is your Help Desk for all questions related to BioTek product use, maintenance, troubleshooting support and other frequently asked questions. 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