Using the Cytation 5 cabinet Imaging Microplate leader to screen Cell culture Status

Author: Paul Held, Ph.D.,Laboratory Manager, Applications Department, civicpride-kusatsu.net Instruments, Inc., Winooski, VT USA

Abstract

Media formulations because that the propagation the in vitro tissue culture often save the pH indicator dye phenol red. This dye has actually long been provided to quickly assess the condition of bulk cultures being propagated because that subsequent speculative analysis. However, as soon as an really experiment has actually been initiated, the state that phenol red in the media is generally no longer utilized, specifically with image-based proliferation assays. Here we define the usage of the Cytation™ 5 cell Imaging Microplate leader to monitor pH, via absorbance measurements, in tissue culture media.

You are watching: Why does phenol red turn yellow

Introduction

There are many formulations for in vitro cell propagation. Relying on the intended cell kind different mixture of salts, amino acids, vitamins, buffers, carbohydrates, fat acids, and also supplements such together serum room used. Countless of these characterized mixtures also contain phenol red. Phenol red has actually been offered as a pH indicator dye in tissue culture media for years (Figure 1). If its existence is not vital for keeping cell cultures, it is frequently used as a quick method for researchers to inspect on culture stocks.

Most mammalian tissues exist at a close to neutral pH. Human being arterial blood is kept at 7.4 (7.35-7.45) by method of a bicarbonate buffers mechanism regulated v normal human body respiration. Deviations native the normal variety induce the body to rise or to decrease lung task in bespeak to alter CO2 expiration<1>. Not surprisingly in vitro farming of cells and also tissues prosper at the very same pH levels. Cultures are kept at physiological pH primarily by two different buffer systems. Bicarbonate-CO2 equipment use a suitable concentration of liquified bicarbonate with man-made levels the carbon dioxide gas. Carbon dioxide dissolves into the media developing carbonic mountain as the reacts through water. Carbonic acid and also bicarbonate also interact to form an equilibrium that is maybe to preserve pH at physiological levels.

*

Figure 1. common Tissue culture Media with Phenol red.

In addition to bicarbonate systems, the most frequently utilized alternate is HEPES buffer, i beg your pardon was an initial described by Good, et al. (1966)<2>. This acts as a zwitterion and also has proven exceptional to conventional buffers in comparative biological assays through cell-free preparations. That has many properties which make it right as a buffer to tissue culture media, principally in the it does not need an enriched setting to keep the exactly pH. Note that its visibility as a buffer certified dealer does not fully eliminate the need for bicarbonate in media formulations.

*

Figure 2. framework of Phenol red and different pH levels. With raising pH phenol red transforms from a zwitter ion to an anion structure and also eventually a di-anion. In law so, the color of the compound transforms from yellow come red come fuchsia.

Regardless the the buffering agent employed, tissue society media is regularly supplemented v phenol red dye. When phenol red has been explained as a weak estrogen under part conditions<4> it is for the most part an inert compound added to in vitro culture media together a visual pH indicator. While concentrations differ with different media formulations, when present it is typically in the 5-15 mg/mL range.

Phenol red, additionally known together phenolsulfonphthalein, is a pH indicator dye the exhibits a gradual transition from yellow to red over a pH range of 6.2 come 8.2 (Figure 2). Above 8.2 the dye turns a shining fuchsia color. In systems at very low pH phenol red is colorless and exists together a zwitterion, include both a negatively charged sulfate group and also a positively fee ketone group. With boost in pH (pKa=1.2) the excess proton in the ketone team is lost, resulting in a yellow color. Additional increases in pH (pKa=7.7) reasons a ns of the hydrogen indigenous the hydroxyl group resulting in a red color. The ratio of these three moieties allows phenol red come be used as a pH indicator dye<3>.

Waste commodities produced by dying cells or overgrowth of contaminants will cause a decrease in pH, bring about a change in indicator color. Because that example, pollution of a society of relatively slowly dividing mammalian cells deserve to be conveniently overgrown by bacteria, causing the acidification of the medium, and also the indicator transforming yellow. Mammalian cell waste commodities themselves will progressively decrease the pH, gradually transforming the systems orange and also then yellow. This color change is an indication that even in the lack of contamination, the media requirements replacing.

Most cellular experiment focus completely on the biology in ~ hand. Even if it is the results are fluorescent, luminescent or image-based, the lift milieu of the media is normally ignored. Here we describe the use of the Cytation™ 5 cell Imaging Multi-Mode reader to screen cell culture pH standing using absorbance-based measurements.

Materials and also Methods

DMEM, RPMI 1640, and also McCoys 5A were purchased indigenous Life Technologies. Phenol red flour (cat # P-3532) to be from Sigma Aldrich. Spectral analysis was determined in 1 nm increments utilizing a Cytation 5 cell Imaging Multi-Mode Reader and a Hellma quartz 96-well microplate. Spectra were established in 1 nm increments utilizing a Cytation 5 cabinet Imaging Multi-Mode Reader.

For pH studies, finish tissue society media include 10% Fetal Bovine serum (800 μL) was diluted with a series of 100 mM buffers (400 μL) at different pH levels. Because that pH levels from 4-8 phosphate buffer to be used, when borate buffer was supplied at pH levels above 8.0 Buffer stocks were all set previously and also the pH determined using one Orion 3 Star pH meter.

Live cell Experiments

NIH3T3 fibroblasts and also were cultured in advanced DMEM supplemented through 10% fetal bovine serum and penicillin-streptomycin at 37 °C in 5% CO2. Cultures were consistently trypsinized (0.05% Trypsin-EDTA) in ~ 80% confluence. For experiments, cells to be plated right into Corning 3904 black color sided clean bottom 96-well microplates. Humidified growth measurements were performed making use of a Cytation 5 cabinet Imaging Multi-Mode leader interfaced v BioSpa™ 8 automatically Incubator (civicpride-kusatsu.net tools Winooski, VT). The BioSpa system controls reader scheduling and also maintains cells in a humidified managed environment (37 °C, 5% CO2) in in between imaging and plate absorbance measurements. As required, the BioSpa transports a microplate come the Cytation 5 because that imaging and also absorbance measurements and returns it to the incubator afterward. Non-humidified experiments were performed directly in a Cytation 5 cabinet Imaging Multi-Mode Reader offered with 5% CO2 or no as indicated. The key was preserved at 37 °C v a 2 °C gradient optimal to bottom to stop condensation from creating on the top lid. In part experiments, a Breath basic gas permeable membrane was provided to covering the wells.

Cells were seeded in ~ a thickness of 4000 cells per well and enabled 24 hours for attachment and also used the following day cells for testing. The result of carbon dioxide gas loss was tested using absorbance measurements every 30 minutes for 12 hours. Comparison were made in between plates that were kept in a 5% CO2 setting with plates the did not. In order to check the result of contamination, cells to be inoculated v either yeast (S. Cerevisiea) or bacteria (E. Coli) diluted in media. Absorbance measurements were performed on 96-well plate societies every hour because that 8 hours using a Cytation 5 cabinet Imaging Multi-Mode Reader. In between absorbance measurements, bowl were positioned in a BioSpa 8, which maintained cells at 37 °C in a humidified 5% CO2 atmosphere. PH levels were figured out by interpolating a previously created calibration curve with the 560 absorbance values.

Results

The absorbance spectra the DMEM media and also phenol red dissolved in PBS to be determined and also compared. Together observed in number 3, complete media has actually a huge peak in absorbance in the UV variety and two lesser peaks focused on 415 nm and 560 nm. This lesser peaks exchange mail to peaks seen v phenol red only solution. Together the concentration the phenol red systems was equivalent to the reported cooking recipes of the media formulation, the very near match in the shape and height that the peaks centered on 415 nm and 560 nm suggests that this peaks represent phenol red absorbance in media. The large UV-peak is most likely because of the huge amount that protein, flavonoids, vitamins, and also nucleosides existing in cell culture media formulations.

*

Figure 3. Comparison the phenol red and compete DMEM media absorbance spectra. The absorbance spectra the phenol red (15 mg/L) dissolved in PBS (pH 7.4) and also complete DMEM comprise 10% FBS were determined. Data represents the mean of three spectral curves after lift subtraction.

The pH of complete media was differed from 4.5 come 9.0 utilizing phosphate buffer. At high pH the media has a clearly shows fuchsia color that transforms to red then yellow together the pH deceases. This color readjust is reflected in the changes in the wavelength peaks in ~ 560 and 415 nm. As watched in number 4, the absorbance optimal at 560 nm is really pronounced at pH 9.0, but almost disappears as soon as the pH is reduced to 4.5. A smaller top is it was observed at 415 nm v acidic pH levels that diminishes with boost in pH.

*

Figure 4. Absorbance Spectra of complete DMEM culture media at various pH levels. Wavelength with an altering peak values v pH are indicated with arrows. Data represents the average of 3 spectral curves for each pH.

Closer check of the 2 absorbance peaks in media with and without phenol red expose an influence from the media constituents to the absorbance in ~ 415 nm (Figure 5), vice versa, the more substantial top at 560 nm seems to no be impacted by media components. This argues that utilizing the 560 nm optimal would be much more advantageous for direct calculation that pH. Using a 415/560 ratiometric analysis will administer a greater dynamic change, yet results in much less accurate pH determination.

*

Figure 5. Absorbance spectra of DMEM culture media with and without phenol red. Wavelength through absorbance height in the visibility (+) or lack (-) the phenol red is indicated with one arrow. Data to represent the average of two spectral curves for each.

The values of the 560 nm absorbance optimal at various pH levels deserve to be plotted and used as a calibration curve because that subsequent long term live-cell assays. As shown in number 6, the plot displayed a linear increase of absorbance from about pH 6.5 (~ 0.1 OD) to 10 (~ 1.1 OD).

*

Figure 6. Absorbance at 560 nm for complete Media at miscellaneous pH levels. The absorbance of finish DMEM media to be measured in ~ 560 nm at miscellaneous pH levels and also plotted as a role of pH. Data to represent the mean and standard deviation of 4 data points.

Despite the inaccuracies the the 415 nm measurement, the ratio of the 415 and 560 nm absorbance values provides expanded coverage in the acidic an ar of pH whereby the 560 nm measurement loses sensitivity. As demonstrated in figure 7, the linear portion of the plot extends indigenous pH 5 (ratio ~ 8) to pH 7.5 (ratio ~ 1).

*

Figure 7. Ratio of 415 and 560 nm absorbance values for complete DMEM media at assorted pH Levels. The absorbance of complete DMEM media was measured in ~ 415 nm and 560 nm at assorted pH levels and the ratio of the 2 values plotted together a duty of pH. Data represents the mean and also standard deviation of three data points.

In bespeak to properly be supplied as a way to determine pH in genuine time, it is important to be able to correct for any background absorbance resulting from the bowl itself. Typically, this is accomplished by either dedicating details blank wells on an speculative plate or making use of a wavelength no effected experimentally and subtracting its worth from the outcomes as the an initial calculation.

Figure 8 demonstrates that using the absorbance in ~ 750 nm is a viable choice for lift subtraction. Spectral data shows that phenol red does not have significant absorbance at the wavelength (data not shown). The resultant values utilizing this an approach differ just slightly from devoted blank-well subtraction at an extremely high pH. This method of correction has the included benefit the correcting for particular well-to-well variances, whereas defined blanks can only accommodate an average plate background.

*

Figure 8. To compare of Background individually Methods. The absorbance of complete DMEM media to be measured at 560 nm at miscellaneous pH levels using either devoted blank wells with PBS buffer or the absorbance at 750 nm come subtract elevator absorbance. Data to represent the mean and also standard deviation that eight data points.

Absorbance data at 560 nm obeys Beers regulation in regards come pathlength. As displayed in figure 9. Different sample volumes show linearity v respect come volume added to wells that a microplate at all pH level tested. No surprisingly, slope of absorbance for the highest pH was the greatest.

*

Figure 9. Result of pH top top Linearity of 560 nm absorbance with various fluid volumes in every well. Different volumes of DMEMphosphate buffer mixture were aliquoted and the absorbance in ~ 560 nm determined. Data represents the mean and also standard deviation that duplicate data points.

Blanked absorbance analysis of phenol red in media can be carry out using any media formulation that contains the dye. Once the 560 nm absorbance at different pH levels for DMEM, McCoy’s 5A and also RPMI 1640 were examined, all 3 had marked increases with lessened pH. The level of adjust is straight proportional to the lot of phenol red in the media. RPMI 1640, which includes 5 mg/L phenol red had a 560 absorbance 0.342 at a pH that 9.85, while DMEM, which consists of 15 mg/L returned a value of 1.038. McCoys 5A media, which has actually a concentration the 10 mg/L to be intermediate in regards to its solution and phenol red totally free DMEM had actually virtually no absorbance in ~ 560 nm. All three phenol red comprise media formulations had actually no more increases in 560 nm absorbance over 10.5 (Figure 10).

*

Figure 10. Absorbance at 560 nm for various media formulations. Various media formulations to be treated to alter their pH and also equal quantities (200 μL to be aliquoted right into a Corning 3904 microplate and also the absorbance at 560 nm determined. The absorbance values were plotted versus pH. Data to represent the mean and also standard deviation the duplicates.

The pH condition of live cells can be monitored utilizing phenol red absorbance. A usual problem with live cell experiments is the interruption of carbon dioxide gas supply to the experimental incubator. Because the pH that the tissue culture media is based on an equilibrium between dissolved CO2 and also bicarbonate ion, the outgassing of liquified CO2 from the media conveniently results in rise in pH. This is materialized in rise in absorbance at 560 nm. As shown in figure 11, DMEM, McCoys 5A, and also RPMI 1640 media rise in absorbance gradually as dissolved CO2 outgasses when the carbon dioxide gas is no longer existing in the neighboring environment. Note that the raw increase in worth is proportional come the quantity of phenol red present in the formulation. DMEM, which has the many phenol red dye had actually the greatest change, yet the family member fold-increase because that the different media is the exact same (approx. 2x).

*

Figure 11. adjust in absorbance after ~ cessation the CO2 it is provided with various media formulations. Deferent media formulations were aliquoted (200 μL) into wells the a microplate and also equilibrated in a CO2 incubator at 37 °C because that 4 hours. The microplate to be then placed in microplate reader and also the absorbance in ~ 560 nm measured every 30 minute at 37 °C there is no CO2. Data points to represent the typical of 32 determinations.

The degree of pH increase can be figured out by interpolating data from calibration curves. As presented in figure 12, tissue society media rapidly increases in pH by around 2 pH devices in the lack of carbon dioxide gas end time. Media formulations are developed for details CO2 concentrations. Because these formulations room designed to keep physiological pH in the existence of 5% CO2 gas levels, the pH adjust for this formulations are similar.

*

Figure 12. change in pH levels with DMEM after ns of CO2 supply. DMED, McCoys 5A, and RPMI 1640 media were aliquoted (200 μL) right into wells the a microplate and also equilibrated in a CO2 incubator in ~ 37 °C for 4 hours. The microplate to be then put in microplate reader and the absorbance in ~ 560 nm measure up every 30 minutes at 37 °C without CO2. PH worths were identified by interpolating the 560 absorbance values through a previously produced calibration curve. Data points represents the mean of 32 determinations.

Similar outcomes are observed once live cells are current in a microplate that has actually been subjected to a lose of carbon dioxide gas. Together observed in figure 13, societies placed in an setting without carbon dioxide quickly outgas dissolved CO2 and the pH rises. This quick rate the pH increase have the right to somewhat be ameliorated through the usage of a gas permeable membrane. While these membranes are mostly intended to reduce evaporative loss in a dried environment, additionally serve to slow outgassing. Plates preserved in either a dried or humidified a CO2 setting maintain your pH.

*

Figure 13. adjust in pH levels through DMEM after loss of CO2 supply. Change in pH in live cell societies with and also without CO2 loss end time. NIH3T3 cells in were enabled to affix overnight in a humidified 5% CO2 incubator maintained at 37 °C. The following day different microplates were then inserted in microplate reader and the absorbance in ~ 560 nm measure every 30 minute at 37 °C through or without CO2. PH values were identified by interpolating the 560 absorbance values with a previously created calibration curve. Data points represents the average of 96 determinations.

Changes in biological task within the well will certainly also an outcome in measureable transforms in pH. Microbial pollution of mammalian tissue culture often outcomes in a rapid expansion of the microbe, i beg your pardon find very favorable growth conditions. Societies that have actually been contaminated through microbes will have actually a quick decrease in pH because of the high metabolic task present. As displayed in number 14, once mammalian tissue cultures are inoculated through either bacteria or yeast a significant and fast drop in pH, as measured through 560 nm absorbance, deserve to be observed.

*

Figure 14. adjust in pH with culture contamination. NIH3T3 cells were seeded at a thickness of 4000 cells per well. After ~ 24 hrs to allow for attachment, cells were inoculated v either yeast or bacterial. Absorbance dimensions were carry out on 96-well plate societies every hour for 8 hours and the outcomes plotted. PH was determined by interpolating data from a previously created calibration curve. Data to represent the mean and also SEM for 96 determinations at every data point.

Discussion

These data show that the Cytation™ 5 cell Imaging Multi-Mode leader is capable of security cell society pH in live cabinet experiments. Cell societies normally come to be acidic early to boost in cabinet numbers and cellular respiration, bring about a yellowing in color of media formulations containing phenol red. When the readjust in pH for short-term experiments is frequently negligible, with irreversible live cabinet experiments increasing cell numbers and also the longer duration deserve to overwhelm the buffering capacity of the media formulation. The capability to monitor changes in culture pH this in genuine time can permit the researcher to have confidence in the observed speculative results or abort experiment that have actually deleterious pH conditions.

The 560 nm optimal was found it to it is in the most reliable for pH determinations. Changes in absorbance at this wavelength deserve to be provided to monitor cell societies in regards to media pH. Determinations utilizing this wavelength obey Beers legislation with respect come pathlength and also as such deserve to be highly quantitative. The an answer of the 560 nm peaks is specifically sensitive to upwards changes, usually the an outcome of lose of CO2 in the incubation chamber. Rise in pH due to lack that CO2 exposure would be it was observed as an increase in 560 nm absorbance, which have the right to increase 5 fold v a pH change from 7 to 9 through DMEM media.

Alternatively, the ratio of the 415 nm and also 560 nm peaks can also be provided as a marker because that pH change. This analysis is more qualitative than direct 560 nm absorbance, yet demonstrates one extended selection to lower pH values contrasted to the 560 nm measurement. This permits one to screen both cabinet overgrowth and also bacterial contamination. While the is less effective in discerning rise in pH, normally resulting indigenous a cessation of CO2 supply, this phenomenon is usually due to mechanical failure rather than a readjust in true biology.

The usage of a 2nd wavelength to one of two people correct because that plate absorbance or carry out a ratiometric determination has actually the included benefit in that both will certainly correct because that condensation or bubbles in the well that may type over time. The addition of fluid reagents has the potential to create a small air bubble as the an outcome of surface tension. An in similar way the carry of a plate to and also from the reader has the potential to an outcome in condensation of the under surface of the key lid. Both of these phenomena an outcome in boost in measure up absorbance unrelated to true changes in the phenol red absorbance in ~ 560 nm. These rises are consistent throughout the spectrum and as such, ratiometric analysis or lift subtraction will certainly be corrective.

Tissue culture media formulations vary through respect to the phenol red concentration. With transforms in pH, formulations with higher concentrations exhibition a more pronounced change in 560 nm absorbance as contrasted to those with reduced concentrations, which often tends to do them much more sensitive come pH change. However, the fold readjust in the 415/560 ratio is higher in formulations through low phenol red loved one to high concentration formulations and the readjust is many pronounced v a decrease in pH. This make the ratiometric an approach desirable as a means to about assess cultures and also identify contamination. Note that the solution of various media formulations will be different relying on the formulation. Because that example, a 415/560 proportion exceeding 3 with DMEM would certainly be indicative of one acidic environment, when the same ratio in McCoy’s 5A media would need a proportion of about 8 to suggest the same pH change. Regardless of the media formulation, one have to expect a gradual change in pH v time. Even live non-dividing cells are still respiring while not increasing in cell number. A very rapid color readjust would it is in indicative that some type of microbial contamination.

See more: How To Set Time On Baby G Shock, How To Set The Time On A Baby G Watch: 10 Steps

The Cytation™ 5 is an ideal platform to monitor phenol red absorbance adjust with live cell imaging experiments. The leader is distinctive in the it has actually the ability of both the absorbance dimensions using a committed UV-vis monochromator and microscopic imaging utilizing a 6-position target turret and LED irradiate cubes. The quick speed of the absorbance analysis adds just seconds to a full 96-well imaging step, yet can provide effective information about cell society status. Gen5™ Microplate Reader and also Imager Software, besides regulating reader function, have the right to be used to calculation pH from previously developed pH calibration curves. This unique mix allows consistent real-time monitoring of long-term live cell society experiments.