What Caused The Phenol Red Solution To Change Colors? What Processes Caused This Change?

Using the Cytation v Cell Imaging Microplate Reader to Monitor Jail cell Culture Status

Writer: Paul Held, Ph.D.,Laboratory Managing director, Applications Department, BioTek Instruments, Inc., Winooski, VT The states

Abstract

Media formulations for the propagation of in vitro tissue culture often contain the pH indicator dye phenol ruddy. This dye has long been used to rapidly appraise the status of majority cultures being propagated for subsequent experimental analysis. However, once an actual experiment has been initiated, the land of phenol blood-red in the media is typically no longer utilized, particularly with paradigm-based proliferation assays. Hither we draw the employ of the Cytation™ 5 Jail cell Imaging Microplate Reader to monitor pH, via absorbance measurements, in tissue culture media.

Introduction

At that place are numerous formulations for in vitro cell propagation. Depending on the intended prison cell type different mixtures of salts, amino acids, vitamins, buffers, carbohydrates, fatty acids, and supplements such equally serum are used. Many of these defined mixtures also comprise phenol red. Phenol ruby has been used as a pH indicator dye in tissue culture media for decades (Figure 1). While its presence is not critical for maintaining cell cultures, information technology is often used as a quick means for researchers to bank check on civilisation stocks.

Most mammalian tissues exist at a virtually neutral pH. Homo arterial blood is maintained at 7.4 (7.35-7.45) by mode of a bicarbonate buffers system regulated through normal torso respiration. Deviations from the normal range induce the body to increase or decrease lung activity in guild to alter CO₂ expiration^[1]. Not surprisingly in vitro cultivation of cells and tissues prosper at the aforementioned pH levels. Cultures are maintained at physiological pH primarily past two different buffer systems. Bicarbonate-CO_ii systems use a matched concentration of dissolved bicarbonate with artificial levels of carbon dioxide gas. Carbon dioxide dissolves into the media forming carbonic acid as it reacts with water. Carbonic acrid and bicarbonate likewise collaborate to form an equilibrium that is able to maintain pH at physiological levels.

Figure one. Typical Tissue Culture Media with Phenol ruddy.

In addition to bicarbonate systems, the virtually commonly utilized alternative is HEPES buffer, which was first described by Good, et al. (1966)^[two]. This acts as a zwitterion and has proven superior to conventional buffers in comparative biological assays with jail cell-free preparations. It has many backdrop which make it ideal as a buffer to tissue civilization media, principally in that information technology does not crave an enriched atmosphere to maintain the correct pH. Note that its presence equally a buffer agent does not completely eliminate the need for bicarbonate in media formulations.

Figure 2. Structure of Phenol red and unlike pH levels. With increasing pH phenol reddish changes from a zwitter ion to an anion structure and somewhen a di-anion. In doing and then, the color of the compound changes from yellowish to ruddy to fuchsia.

Regardless of the buffering amanuensis employed, tissue culture media is frequently supplemented with phenol red dye. While phenol reddish has been described every bit a weak estrogen under some conditions^[4] information technology is for the near part an inert compound added to in vitro culture media equally a visual pH indicator. While concentrations vary with different media formulations, when nowadays information technology is typically in the 5-15 mg/mL range.

Phenol cherry-red, also known every bit phenolsulfonphthalein, is a pH indicator dye that exhibits a gradual transition from xanthous to red over a pH range of half-dozen.two to 8.2 (Figure 2). To a higher place eight.two the dye turns a bright fuchsia color. In solution at very low pH phenol red is colorless and exists equally a zwitterion, containing both a negatively charged sulfate grouping and a positively charged ketone grouping. With an increase in pH (pKa=ane.2) the excess proton in the ketone group is lost, resulting in a yellow color. Further increases in pH (pKa=7.seven) causes a loss of the hydrogen from the hydroxyl group resulting in a red color. The ratio of these three moieties enables phenol red to be used as a pH indicator dye^[3].

Waste material products produced by dying cells or overgrowth of contaminants will crusade a subtract in pH, leading to a change in indicator color. For instance, contamination of a culture of relatively slowly dividing mammalian cells can exist quickly overgrown by bacteria, resulting in the acidification of the medium, and the indicator turning yellow. Mammalian cell waste products themselves will slowly decrease the pH, gradually turning the solution orange and then yellow. This color alter is an indication that even in the absence of contagion, the media needs replacing.

Most cellular experiments focus entirely on the biology at hand. Whether the results are fluorescent, luminescent or image-based, the groundwork milieu of the media is generally ignored. Here we describe the utilize of the Cytation™ 5 Cell Imaging Multi-Manner Reader to monitor jail cell civilisation pH status using absorbance-based measurements.

Materials and Methods

DMEM, RPMI 1640, and McCoys 5A were purchased from Life Technologies. Phenol red pulverisation (cat # P-3532) was from Sigma Aldrich. Spectral analysis was adamant in 1 nm increments using a Cytation 5 Prison cell Imaging Multi-Fashion Reader and a Hellma quartz 96-well microplate. Spectra were determined in 1 nm increments using a Cytation 5 Cell Imaging Multi-Mode Reader.

For pH studies, consummate tissue culture media containing 10% Fetal Bovine serum (800 μL) was diluted with a series of 100 mM buffers (400 μL) at different pH levels. For pH levels from 4-8 phosphate buffer was used, while borate buffer was used at pH levels higher up viii.0 Buffer stocks were prepared previously and the pH determined using an Orion 3 Star pH meter.

Live Cell Experiments

NIH3T3 fibroblasts and were cultured in Advanced DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37 °C in v% CO_two. Cultures were routinely trypsinized (0.05% Trypsin-EDTA) at fourscore% confluence. For experiments, cells were plated into Corning 3904 black sided clear bottom 96-well microplates. Humidified growth measurements were performed using a Cytation 5 Cell Imaging Multi-Mode Reader interfaced with BioSpa™ 8 Automated Incubator (BioTek Instruments Winooski, VT). The BioSpa organization controls reader scheduling and maintains cells in a humidified controlled environment (37 °C, 5% CO_ii) in between imaging and plate absorbance measurements. As required, the BioSpa transports a microplate to the Cytation 5 for imaging and absorbance measurements and returns it to the incubator subsequently. Not-humidified experiments were performed straight in a Cytation 5 Jail cell Imaging Multi-Fashion Reader supplied with 5% CO₂ or non as indicated. The plate was maintained at 37 °C with a 2 °C gradient top to lesser to preclude condensation from forming on the top lid. In some experiments, a Jiff Easy gas permeable membrane was used to embrace the wells.

Cells were seeded at a density of 4000 cells per well and allowed 24 hours for zipper and used the post-obit mean solar day cells for testing. The effect of carbon dioxide gas loss was tested using absorbance measurements every 30 minutes for 12 hours. Comparison were made between plates that were maintained in a 5% CO₂ environment with plates that did not. In lodge to test the upshot of contamination, cells were inoculated with either yeast (S. cerevisiea) or bacteria (Due east. coli) diluted in media. Absorbance measurements were performed on 96-well plate cultures every hour for eight hours using a Cytation 5 Cell Imaging Multi-Mode Reader. Between absorbance measurements, plates were situated in a BioSpa 8, which maintained cells at 37 °C in a humidified 5% CO₂ temper. pH levels were adamant by interpolating a previously generated calibration curve with the 560 absorbance values.

Results

The absorbance spectra of DMEM media and phenol red dissolved in PBS were determined and compared. Equally 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. These lesser peaks stand for to peaks seen with phenol cherry just solution. Equally the concentration of phenol red solution was equivalent to the reported recipe of the media conception, the very near match in the shape and height of the peaks centered on 415 nm and 560 nm suggests that these peaks represent phenol red absorbance in media. The large UV-pinnacle is most probable due to the large amount of protein, flavonoids, vitamins, and nucleosides present in cell culture media formulations.

Figure 3. Comparison of phenol cherry-red and compete DMEM media absorbance spectra. The absorbance spectra of phenol cherry (fifteen mg/L) dissolved in PBS (pH vii.4) and complete DMEM containing x% FBS were determined. Data represents the mean of three spectral curves after background subtraction.

The pH of consummate media was varied from 4.5 to 9.0 using phosphate buffer. At high pH the media has a visible fuchsia color that changes to blood-red then yellow every bit the pH deceases. This color change is reflected in the changes in the wavelength peaks at 560 and 415 nm. As seen in Figure 4, the absorbance acme at 560 nm is very pronounced at pH 9.0, but near disappears when the pH is decreased to 4.5. A smaller peak is observed at 415 nm with acidic pH levels that diminishes with an increment in pH.

Figure 4. Absorbance Spectra of consummate DMEM civilization media at different pH levels. Wavelengths with changing pinnacle values with pH are indicated with arrows. Data represents the average of 3 spectral curves for each pH.

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 v), whereas the more than substantial peak at 560 nm seems to not exist afflicted past media components. This suggests that using the 560 nm peak would be more advantageous for direct calculation of pH. Using a 415/560 ratiometric analysis will provide a greater dynamic change, but results in less accurate pH conclusion.

Figure five. Absorbance spectra of DMEM civilization media with and without phenol red. Wavelength with absorbance peak in the presence (+) or absence (-) of phenol carmine is indicated with an arrow. Data represents the average of 2 spectral curves for each.

The values of the 560 nm absorbance peak at different pH levels can be plotted and used as a scale bend for subsequent long term live-cell assays. Equally shown in Figure 6, the plot demonstrates a linear increment of absorbance from about pH half dozen.5 (~ 0.i OD) to 10 (~ i.i OD).

Effigy half dozen. Absorbance at 560 nm for Complete Media at Various pH levels. The absorbance of complete DMEM media was measured at 560 nm at diverse pH levels and plotted as a function of pH. Data represents the mean and standard departure of iv data points.

Despite the inaccuracies of the 415 nm measurement, the ratio of the 415 and 560 nm absorbance values provides extended coverage in the acidic region of pH where the 560 nm measurement loses sensitivity. As demonstrated in Effigy 7, the linear portion of the plot extends from pH 5 (ratio ~ 8) to pH 7.5 (ratio ~ 1).

Figure seven. Ratio of 415 and 560 nm absorbance values for Complete DMEM media at various pH Levels. The absorbance of complete DMEM media was measured at 415 nm and 560 nm at diverse pH levels and the ratio of the two values plotted as a function of pH. Information represents the mean and standard deviation of 3 data points.

In gild to effectively be used as a means to make up one's mind pH in real time, it is necessary to be able to correct for any background absorbance resulting from the plate itself. Typically, this is achieved by either dedicating specific blank wells on an experimental plate or using a wavelength non effected experimentally and subtracting its value from the results every bit the starting time adding.

Figure eight demonstrates that using the absorbance at 750 nm is a viable option for background subtraction. Spectral data indicates that phenol red does not accept pregnant absorbance at that wavelength (data not shown). The resultant values using this method differ merely slightly from dedicated blank-well subtraction at very high pH. This method of correction has the added do good of correcting for specific well-to-well variances, whereas defined blanks can merely adapt an boilerplate plate background.

Figure 8. Comparison of Background Subtraction Methods. The absorbance of complete DMEM media was measured at 560 nm at diverse pH levels using either dedicated blank wells with PBS buffer or the absorbance at 750 nm to subtract background absorbance. Information represents the mean and standard deviation of eight data points.

Absorbance data at 560 nm obeys Beers law in regards to pathlength. Every bit shown in Figure nine. Different sample volumes show linearity with respect to volume added to wells of a microplate at all pH levels tested. Not surprisingly, slope of absorbance for the highest pH was the greatest.

Figure 9 . Upshot of pH on Linearity of 560 nm absorbance with unlike fluid volumes in each well. Unlike volumes of DMEMphosphate buffer mixture were aliquoted and the absorbance at 560 nm determined. Data represents the mean and standard deviation of duplicate data points.

Blanked absorbance analysis of phenol ruby-red in media can be performed using any media formulation that contains the dye. When the 560 nm absorbance at different pH levels for DMEM, McCoy's 5A and RPMI 1640 were examined, all 3 had marked increases with reduced pH. The caste of change is direct proportional to the amount of phenol cherry-red in the media. RPMI 1640, which contains 5 mg/L phenol reddish had a 560 absorbance 0.342 at a pH of ix.85, while DMEM, which contains 15 mg/L returned a value of 1.038. McCoys 5A media, which has a concentration of x mg/L was intermediate in regards to its response and phenol reddish free DMEM had virtually no absorbance at 560 nm. All three phenol red containing media formulations had no farther increases in 560 nm absorbance above 10.5 (Figure ten).

Effigy ten. Absorbance at 560 nm for different media formulations. Different media formulations were treated to alter their pH and equal volumes (200 μL were aliquoted into a Corning 3904 microplate and the absorbance at 560 nm adamant. The absorbance values were plotted against pH. Data represents the mean and standard deviation of duplicates.

The pH status of live cells can be monitored using phenol crimson absorbance. A common problem with live jail cell experiments is the interruption of carbon dioxide gas supply to the experimental incubator. Because the pH of the tissue culture media is based on an equilibrium between dissolved CO₂ and bicarbonate ion, the outgassing of dissolved CO₂ from the media chop-chop results in an increase in pH. This is manifested in an increase in absorbance at 560 nm. As shown in Effigy 11, DMEM, McCoys 5A, and RPMI 1640 media increase in absorbance over time as dissolved CO_ii outgasses when the carbon dioxide gas is no longer present in the surrounding environs. Note that the raw increment in value is proportional to the amount of phenol ruddy present in the formulation. DMEM, which contains the well-nigh phenol scarlet dye had the greatest modify, but the relative fold-increment for the dissimilar media is the aforementioned (approx. 2x).

Effigy 11. Change in absorbance subsequently cessation of CO_two supply with unlike media formulations. Deferent media formulations were aliquoted (200 μL) into wells of a microplate and equilibrated in a CO₂ incubator at 37 °C for four hours. The microplate was then placed in microplate reader and the absorbance at 560 nm measured every xxx minutes at 37 °C without CO_two. Data points represents the mean of 32 determinations.

The extent of pH increase can be determined by interpolating information from scale curves. Equally shown in Effigy 12, tissue culture media quickly increases in pH by approximately ii pH units in the absence of carbon dioxide gas over time. Media formulations are developed for specific CO₂ concentrations. Because these formulations are designed to maintain physiological pH in the presence of v% CO₂ gas levels, the pH change for these formulations are similar.

Figure 12. Alter in pH levels with DMEM after loss of CO_two supply. DMED, McCoys 5A, and RPMI 1640 media were aliquoted (200 μL) into wells of a microplate and equilibrated in a CO₂ incubator at 37 °C for 4 hours. The microplate was so placed in microplate reader and the absorbance at 560 nm measured every thirty minutes at 37 °C without CO₂. pH values were adamant by interpolating the 560 absorbance values with a previously generated calibration curve. Data points represents the mean of 32 determinations.

Similar results are observed when alive cells are present in a microplate that has been subjected to a loss of carbon dioxide gas. Equally observed in Figure 13, cultures placed in an environs without carbon dioxide quickly outgas dissolved CO_two and the pH rises. This rapid rate of pH increase can somewhat exist ameliorated by the use of a gas permeable membrane. While these membranes are primarily intended to reduce evaporative loss in a dry environment, also serve to dull outgassing. Plates maintained in either a dry or humidified a CO₂ environment maintain their pH.

Effigy 13. Change in pH levels with DMEM later loss of CO₂ supply. Change in pH in live cell cultures with and without CO₂ loss over fourth dimension. NIH3T3 cells in were allowed to attach overnight in a humidified 5% CO₂ incubator maintained at 37 °C. The post-obit day separate microplates were and then placed in microplate reader and the absorbance at 560 nm measured every 30 minutes at 37 °C with or without CO₂. pH values were determined by interpolating the 560 absorbance values with a previously generated calibration curve. Data points represents the hateful of 96 determinations.

Changes in biological activity within the well volition also result in measureable changes in pH. Microbial contagion of mammalian tissue culture often results in a rapid growth of the microbe, which observe very favorable growth conditions. Cultures that have been contaminated with microbes will have a rapid decrease in pH due to the high metabolic activity present. As shown in Effigy 14, when mammalian tissue cultures are inoculated with either bacteria or yeast a pregnant and rapid drop in pH, as measured by 560 nm absorbance, can exist observed.

Figure 14. Change in pH with culture contamination. NIH3T3 cells were seeded at a density of 4000 cells per well. After 24 hours to permit for attachment, cells were inoculated with either yeast or bacterial. Absorbance measurements were performed on 96-well plate cultures every hour for eight hours and the results plotted. pH was determined past interpolating data from a previously generated calibration curve. Data represents the mean and SEM for 96 determinations at each information bespeak.

Discussion

These data demonstrate that the Cytation™ 5 Cell Imaging Multi-Manner Reader is capable of monitoring cell civilisation pH in live prison cell experiments. Jail cell cultures normally become acidic due to an increase in prison cell numbers and cellular respiration, resulting in a yellowing in color of media formulations containing phenol red. While the modify in pH for short-term experiments is oftentimes negligible, with long-term alive cell experiments increasing prison cell numbers and the longer duration tin can overwhelm the buffering chapters of the media formulation. The ability to monitor changes in civilization pH this in existent fourth dimension can allow the researcher to have confidence in the observed experimental results or abort experiments that have deleterious pH conditions.

The 560 nm peak was found it to be the about reliable for pH determinations. Changes in absorbance at this wavelength can be used to monitor cell cultures in regards to media pH. Determinations using this wavelength obey Beers law with respect to pathlength and as such tin can be highly quantitative. The response of the 560 nm peaks is particularly sensitive to upward changes, commonly the result of loss of CO_ii in the incubation bedroom. An increase in pH due to lack of CO_ii exposure would exist observed every bit an increase in 560 nm absorbance, which can increment five fold with a pH change from 7 to nine with DMEM media.

Alternatively, the ratio of the 415 nm and 560 nm peaks tin also exist used as a mark for pH modify. This analysis is more than qualitative than direct 560 nm absorbance, but demonstrates an extended range to lower pH values compared to the 560 nm measurement. This allows i to monitor both cell overgrowth and bacterial contagion. While it is less constructive in discerning an increase in pH, usually resulting from a cessation of CO_ii supply, this phenomenon is usually due to mechanical failure rather than a change in truthful biology.

The use of a second wavelength to either correct for plate absorbance or provide a ratiometric conclusion has the added do good in that both volition correct for condensation or bubbling in the well that may grade over time. The addition of liquid reagents has the potential to produce a small air bubble every bit the result of surface tension. Likewise the transfer of a plate to and from the reader has the potential to event in condensation of the under surface of the plate lid. Both of these phenomena upshot in an increase in measured absorbance unrelated to true changes in the phenol carmine absorbance at 560 nm. These increases are consistent beyond the spectrum and as such, ratiometric analysis or background subtraction will be corrective.

Tissue culture media formulations vary with respect to the phenol red concentration. With changes in pH, formulations with higher concentrations exhibit a more pronounced change in 560 nm absorbance as compared to those with lower concentrations, which tends to make them more sensitive to pH modify. However, the fold change in the 415/560 ratio is greater in formulations with depression phenol red relative to high concentration formulations and the change is nearly pronounced with a subtract in pH. This brand the ratiometric method desirable as a means to roughly assess cultures and identify contamination. Note that the response of dissimilar media formulations will exist different depending on the formulation. For example, a 415/560 ratio exceeding 3 with DMEM would be indicative of an acidic surroundings, while the aforementioned ratio in McCoy'southward 5A media would require a ratio of approximately viii to bespeak the same pH change. Regardless of the media formulation, one should expect a gradual alter in pH with time. Even live non-dividing cells are still respiring while non increasing in jail cell number. A very rapid color change would be indicative of some sort of microbial contamination.

The Cytation™ 5 is an ideal platform to monitor phenol carmine absorbance modify with live cell imaging experiments. The reader is unique in that information technology has the adequacy of both the absorbance measurements using a dedicated UV-vis monochromator and microscopic imaging using a 6-position objective turret and LED lite cubes. The rapid speed of the absorbance reading adds only seconds to a full 96-well imaging step, but can provide constructive information regarding cell culture condition. Gen5™ Microplate Reader and Imager Software, also controlling reader office, can be used to calculate pH from previously established pH calibration curves. This unique combination allows continual real-fourth dimension monitoring of long-term live cell civilisation experiments.

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References

1. Rhoades, Rodney A.; Bell, David R. (2012). Medical physiology principles for clinical medicine (fourth ed., International ed.). Philadelphia, Pa.: Lippincott Williams & Wilkins. ISBN 9781451110395.
2. Practiced, N.E., Yard.D. Winger, W. Winter, T.North. Connelly, South. Izawa, and R.K.M. Singh (1966) Hydrogen Ion Buffers for Biological Research, Biochemistry, 5(2):467-477. DOI: 10.1021/bi00866a011
3. Merck Index, 13th ed., 7329 Phenolsulfonphthalein
4. Berthois, Y.; Katzenellenbogen, J. A.; Katzenellenbogen, B. S. (1986). "Phenol red in tissue culture media is a weak estrogen: Implications concerning the written report of estrogen-responsive cells in culture" (pdf). Proceedings of the National University of Sciences of the U.s.. 83 (viii): 2946- 2500. PMC 323325 . PMID 3458212. doi:ten.1073/ pnas.83.8.2496.

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