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I've noticed that in mammalian cell culture, there are often two types of DMEM available. High Glucose and Low Glucose. Does it matter which type I use for culturing of cells (e.g. Hela or HEK293)? Which is the type people normally use?
We typically use High Glucose as cells grow faster. One caveat is that there are concerns that certain protein modifications (specifically OGlnAc-ylation) may be upregulated in non-physiological (25mM or high glucose) media.
HyClone Dulbecco's Modified Eagle Medium (DMEM) with high glucose: Liquid
HyClone DMEM medium promotes cell growth in research and biomanufacturing cell culture processes.
HyClone DMEM high glucose liquid media supports cell growth in research, upstream process development, and cell culture manufacturing processes.
- DMEM high glucose medium is manufactured in cGMP (21 CFR 820) compliant and ISO9001 certified facilities.
- DMEM high glucose medium efficacy and homogeneity is tested with HyClone sera.
DMEM media contain increased concentrations of vitamins, minerals, and amino acids compared to traditional basal media. DMEM medium composition also includes ferric nitrate, making this cell culture medium an option for certain cell lines requiring iron for cell growth. HyClone DMEM media preparations are available in high or low glucose formulations, as well as with or without L-glutamine, magnesium, and sodium pyruvate. DMEM formulation options may also include phenol red as a pH indicator. All available DMEM media products contain a sodium bicarbonate buffer with or without HEPES to maintain optimal culture pH.
HyClone DMEM raw material components are screened through strict quality control testing for lot-to-lot consistency. DMEM liquid media products are hydrated using purified water and undergo sterile filtration.
Eagle's minimal essential medium
Minimal Essential Medium (MEM) is a synthetic cell culture medium developed by Harry Eagle first published in 1959  in Science that can be used to maintain cells in tissue culture. It is based on 6 salts and glucose described in Earle's salts in 1934: (calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium phosphate and sodium bicarbonate), supplemented with 13 essential amino acids, and 8 vitamins: thiamine (vitamin B1), riboflavin (vitamin B2), nicotinamide (vitamin B3), pantothenic acid (vitamin B5), pyrodoxine (vitamin B6), folic acid (vitamin B9), choline, and myo-inositol (originally known as vitamin B8).
Many variations of this medium have been developed, mostly adding additional vitamins, amino acids, and/or other nutrients. 
Eagle developed his earlier "Basal Medium Eagle" (BME) in 1955–1957 on mouse L cells  and human HeLa cells,  with 13 essential amino acids and 9 vitamins added. BME contains biotin (vitamin B7), which Eagle later found to be superfluous. His 1959 "Minimal Essential Medium" doubles the amount of many amino acids to "conform more closely to the protein composition of cultured human cells. This permits the cultures to be kept for somewhat longer periods without refeeding". 
DMEM (Dulbecco's modified Eagle's medium) was originally suggested as Eagle's medium with a 'Fourfold concentration of amino acids and vitamins' by Dulbecco and Vogt published in 1959. The commercial version of this medium has additional modifications detailed below. 
α-MEM (Minimum Essential Medium Eagle - alpha modification) is a medium based on MEM published in 1971 by Clifford P. Stanners and colleagues.  It contains more non-essential amino acids, sodium pyruvate, and vitamins (ascorbic acid (vitamin C), biotin, and cyanocobalamin) compared with MEM. It can also come with lipoic acid and nucleosides.  
Glasgow's MEM (Glasgow Minimal Essential Medium) is yet another modification, prepared by lan MacPherson and Michael Stoker. 
One liter of each medium contains (in milligrams):
|Medium||BME ||MEM ||MEMa |
|L-Tyrosine disodium salt dihydrate||26||52||52|
|Calcium Chloride (CaCl2) (anhyd.)||200||200||200|
|Magnesium Sulfate (MgSO4) (anhyd.)||97.67||97.67||97.67|
|Potassium Chloride (KCl)||400||400||400|
|Sodium Bicarbonate (NaHCO3)||2200||2200||2200|
|Sodium Chloride (NaCl)||6800||6800||6800|
|Sodium Phosphate monobasic (NaH2PO4-H2O)||140||140||140|
- ^ ab EAGLE H (1959). "Amino acid metabolism in mammalian cell cultures". Science. 130 (3373): 432–7. Bibcode:1959Sci. 130..432E. doi:10.1126/science.130.3373.432. PMID13675766.
- Yao, T Asayama, Y (April 2017). "Animal-cell culture media: History, characteristics, and current issues". Reproductive Medicine and Biology. 16 (2): 99–117. doi:10.1002/rmb2.12024. PMC5661806 . PMID29259457.
- EAGLE H (1955). "The specific amino acid requirements of a mammalian cell (strain L) in tissue culture". J Biol Chem. 214 (2): 839–52. PMID14381421.
- EAGLE H (1955). "The specific amino acid requirements of a human carcinoma cell (Stain HeLa) in tissue culture". J Exp Med. 102 (1): 37–48. doi:10.1084/jem.102.1.37. PMC2136494 . PMID14392239.
- DULBECCO R, FREEMAN G (1959). "Plaque production by the polyoma virus". Virology. 8 (3): 396–7. doi:10.1016/0042-6822(59)90043-1. PMID13669362.
- Stanners CP, Eliceiri GL, Green H (1971). "Two types of ribosome in mouse-hamster hybrid cells". Nat New Biol. 230 (10): 52–4. doi:10.1038/newbio230052a0. PMID5279808. CS1 maint: multiple names: authors list (link)
- "α-MEM" (PDF) .
- "Alpha MEM with Nucleosides". Stem Cell Tech.
- "Glasgow's Modified Eagle's Medium 51492C". Sigma-Aldrich . Retrieved 4 November 2018 .
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High Glucose vs Low Glucose DMEM for Cell Culture - Biology
Excess reactive oxygen species (ROS) production is thought to play a key role in the loss of pancreatic β-cell number and/or function, in response to high glucose and/or fatty acids. However, contradictory findings have been reported showing that in pancreatic β cells or insulin-secreting cell lines, ROS are produced under conditions of either high or low glucose. Superoxide production was measured in attached INS1E cells as a function of glucose concentration, by following in real time the oxidation of dihydroethidine. Minimal values of superoxide production were measured at glucose concentrations of 5–20 mM, whereas superoxide generation was maximal at 0–1 mM glucose. Superoxide generation started rapidly (15–30 min) after exposure to low glucose and was suppressed by its addition within minutes. Superoxide was totally suppressed by rotenone, but not myxothiazol, suggesting a role for complex I in this process. Indirect evidence for mitochondrial ROS generation was also provided by a decrease in aconitase activity. Activation of AMPK, a cellular metabolic sensor, and its downstream target ACC by low glucose concentration was largely inhibited by addition of MnTBAP, a MnSOD and catalase mimetic that also totally suppressed superoxide production. Taken together, the data show that low glucose activates AMPK in a superoxide-dependent, AMP-independent way.
► Low but not high glucose generated superoxide in the pancreatic beta cell line INS1. ► The effect of low glucose was rapid and reversible. ► Low glucose-induced superoxide production localized at complex 1. ► Low glucose also induced AMPK activation. ► AMPK activation was ROS-dependent and AMP, LKB1 and CaMKK independent.
3/ Electron Transport System (Electron Transport Chain)
The electron transport system/chain is the third and last stage of cellular metabolism and takes place in the folded, inner membrane of the mitochondria (cristae). This is a particularly important stage given that most of the ATP molecules are produced here.
This stage also includes several important steps of electron transfer that include:
Step 1: In the first step of the electron transport system, the NADH molecules come in contact with enzyme complex 1 which takes electrons from these molecules thus converting them from NADH to NAD+ (6 molecules).
Unlike NADH, FADH has a higher affinity for enzyme complex 2 and thus reacts with the enzyme releasing two electrons (2 FADH molecules release electrons to complex 2). This converts the 2 molecules of FADH to 2 molecules of FAD and protons.
Step 2: In step 2 of cellular metabolism, complex 1 releases the electrons it gained from NADH so that it can move from a high energy to a low energy level. This also causes a pore on the complex to open allowing for protons to be pumped out.
While complex 2 also releases electrons in order to switch to a lower energy state, it does not have pores and therefore protons cannot be pumped out. In step 2, all the electrons released by complex 1 and 2 are taken up by Coenzyme Q (also known as ubiquinone).
Step 3: One of the defining characteristics of Coenzyme Q is the fact that it is mobile and can therefore move within the cristae. This is important in this step as it allows the molecule to move in order to transfer the electrons. In this step, the molecule moves and transfers its electrons to enzyme complex 3.
Step 4: As with the other enzyme complexes in this system, enzyme complex 3 also changes to a higher energy level having received electrons. In this state, it has to release these electrons in order to switch back to a lower energy level.
Again, then, the electrons are transferred to another mobile molecule known as Cytochrome C. Like enzyme complex 1, this complex also has a pore than opens following the release of electrons thus allowing for protons to be pumped out into the intermembrane space.
Step 5: In step 5, the process is again repeated with Cytochrome C releasing the electrons in order to switch to a lower energy level. These electrons are then accepted by enzyme complex 4.
Step 6: In step 6, enzyme complex 4 releases the electrons it received thus moving from a high energy to a lower energy level. Here, however, the electrons released combine with oxygen and protons to produce water. The release of electrons here also causes a pore to open thus allowing protons to be pumped out.
* Up to this step, it's evident that the complexes (as well as other involved molecules) receive and release electrons thus creating a chain of electron transfer.
Here, the complexes switch to a higher energy state each time they receive the electrons and have to release these electrons in order to switch back to a more stable, lower energy level.
As a result of releasing protons into the intermembrane space, there is a high accumulation/concentration of these electrons in this space compared to the mitochondria complex.
Because of this difference, a gradient is created which means that they are promoted to move from the area of high proton concentration (in the intermembrane space) to the area of lower proton concentration (the mitochondria matrix). To do this, the protons move through a special structure known as ATP synthase.
Here, protons pass through the stator part of the structure which allows them to move towards the mitochondria matrix. As they pass through, this causes another part of the structure known as the rotor to start spinning. As it spins with the movement of protons into the matrix, the rotor starts to absorb potential energy.
This energy is then used by another part of the ATP synthase structure (the catalytic knob) to create ATP molecules from phosphates and ADP. The ADP and phosphates are contained in the catalytic knob and are used to produce ATP when potential energy is absorbed in the rotor.
Here, the process used to produce ATP from potential energy created through the movement of protons is known as Oxidative Phosphorylation.
* Under aerobic conditions, the three stages of cellular metabolism produce a total of 36 ATP molecules. Anaerobic conditions result in the production of 2 ATP molecules from glycolysis in particular.
What is a normal blood glucose?
Glucose is the key metabolic substrate for tissue energy production. In the perinatal period the mother supplies glucose to the fetus and for most of the gestational period the normal lower limit of fetal glucose concentration is around 3 mmol/L. Just after birth, for the first few hours of life in a normal term neonate appropriate for gestational age, blood glucose levels can range between 1.4 mmol/L and 6.2 mmol/L but by about 72 h of age fasting blood glucose levels reach normal infant, child and adult values (3.5-5.5 mmol/L). Normal blood glucose levels are maintained within this narrow range by factors which control glucose production and glucose utilisation. The key hormones which regulate glucose homoeostasis include insulin, glucagon, epinephrine, norepinephrine, cortisol and growth hormone. Pathological states that affect either glucose production or utilisation will lead to hypoglycaemia. Although hypoglycaemia is a common biochemical finding in children (especially in the newborn) it is not possible to define by a single (or a range of) blood glucose value/s. It can be defined as the concentration of glucose in the blood or plasma at which the individual demonstrates a unique response to the abnormal milieu caused by the inadequate delivery of glucose to a target organ (eg, the brain). Hypoglycaemia should therefore be considered as a continuum and the blood glucose level should be interpreted within the clinical scenario and with respect to the counter-regulatory hormonal responses and intermediate metabolites.
Protein production by auto-induction in high density shaking cultures
Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).
Steps of the Cori cycle
The analysis of the steps of the Cori cycle is made considering the lactate produced by red blood cells and skeletal muscle cells.
Mature red blood cells are devoid of mitochondria, nucleus and ribosomes, and obtain the necessary energy only by glycolysis. The availability of NAD + is essential for glycolysis to proceed as well as for its rate: the oxidized form of the coenzyme is required for the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate in the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase (EC 184.108.40.206).
Glyceraldehyde 3-phosphate + NAD + → 1,3-Bisphosphoglycerate + NADH + H +
The accumulation of NADH is avoided by the reduction of pyruvate to lactate, in the reaction catalyzed by lactate dehydrogenase (EC 220.127.116.11), where NADH acts as reducing agent.
Pyruvate + NADH + H + → Lactate + NAD +
The skeletal muscle, particularly fast-twitch fibers which contain a reduced number of mitochondria, under low oxygen condition, such as during intense exercise, produces significant amounts of lactate. In fact, in such conditions:
- the rate of pyruvate production by glycolysis exceeds the rate of its oxidation by the citric acid cycle, so that less than 10% of the pyruvate enters the citric acid cycle
- the rate at which oxygen is taken up by the cells is not sufficient to allow aerobic oxidation of all the NADH produced.
And, like in red blood cells, the reaction catalyzed by lactate dehydrogenase, regenerating NAD + , allows glycolysis to proceed.
However, lactate is an end product of metabolism that must be converted back into pyruvate to be used.
The plasma membrane of most cells is freely permeable to both pyruvate and lactate that can thus reach the bloodstream. And, regarding for example the skeletal muscle, the amount of lactate that leaves the cell is greater than that of pyruvate due to the high NADH/NAD + ratio in the cytosol and to the catalytic properties of the skeletal muscle isoenzyme of LDH.
Once into the bloodstream, lactate reaches the liver, which is its major user, where it is oxidized to pyruvate in the reaction catalyzed by the liver isoenzyme of lactate dehydrogenase.
Lactate + NAD + → Pyruvate + NADH + H +
In the hepatocyte, this oxidation is favored by the low NADH/NAD + ratio in the cytosol.
Then, pyruvate enters the gluconeogenesis pathway to be converted into glucose.
Glucose leaves the liver, enters into the bloodstream and is delivered to the muscle, as well as to other tissues and cells that require it, such as red blood cells and neurons, thus closing the cycle.
The enzyme is a tetramer composed of two different types of subunits, designed as:
The H subunit predominates in the heart, whereas the M subunit predominates in the skeletal muscle and liver. Typically, tissues in which a predominantly or exclusively aerobic metabolism occurs, such as the heart, synthesize H subunits to a greater extent than M subunits, whereas tissues in which anaerobic metabolism is important, such as skeletal muscle, synthesize M subunits to a greater extent than H subunits.
The two subunits associate in 5 different ways to form homopolymers, that is, macromolecules formed by repeated, identical subunits, or heteropolymers, that is, macromolecules formed by different subunits. Different LDH isoenzymes have different catalytic properties, as well as different distribution in various tissues, as indicated below:
- H4, also called type 1, LDH1, or A4, a homopolymer of H subunits, is found in cardiac muscle, kidney, and red blood cells
- H3M1, also called type 2, LDH2, or A3B, has a tissue distribution similar to that of LDH1
- H2M2, also called type 3, LDH3, or A2B2, is found in the spleen, brain, white cells, kidney, and lung
- H1M3, also called type 4, LDH4, or AB3, is found in the spleen, lung, skeletal muscle, lung, red blood cells, and kidney
- M4, also called type 5, LDH5, or B4, a homopolymer of M subunits, is found in the liver, skeletal muscle, and spleen.
The H4 isoenzyme has a higher substrate affinity than the M4 isoenzyme.
The H4 isoenzyme is allosterically inhibited by high levels of pyruvate (its product), whereas the M4 isoenzyme is not.
The other LDH isoenzymes have intermediate properties, depending on the ratio between the two types of subunits.
It is thought that the H4 isoenzyme is the most suitable for catalyzing the oxidation of lactate to pyruvate that, in the heart, due to its exclusively aerobic metabolism, is then completely oxidized to CO2 and H2O. Instead, the M4 isoenzyme is the main isoenzyme found in skeletal muscle, most suitable for catalyzing the reduction of pyruvate to lactate, thus allowing glycolysis to proceed in anaerobic conditions.
Other metabolic fates of lactate
From the above, it is clear that lactate is not a metabolic dead end, a waste product of glucose metabolism.
And it may have a different fate from that entering the Cori cycle.
For example, in skeletal muscle during recovery following an exhaustive exercise, that is, when oxygen is again available, or if the exercise is of low intensity, lactate is re-oxidized to pyruvate, due to NAD + availability, and then completely oxidized to CO2 and H20, with a greater production of ATP than in anaerobic condition. In such conditions, the energy stored in NADH will be released, yielding on average 2.5 ATP per molecule of NADH.
In addition, lactate can be taken up by exclusively aerobic tissues, such as heart, to be oxidized to CO2 and H20.
Material and Methods
Requirement of Chemicals
Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technology, USA. Antibiotics solution (penicillin-streptomycin) was purchased from HiMedia, India, while ethylenediaminetetraacetic acid (EDTA) was purchased from Sigma, USA. Sildenafil citrate was purchased from Clearsynth, India. All the other chemicals used in this experiment were analytical grade procured from India.
Human Endothelial Hybrid Cell Line (EA. hy926) was used as a test system in this experiment. The cells were maintained in DMEM growth medium for routine culture supplemented with 10% FBS. Growth conditions were maintained at 37°C, 5%CO2, and 95% humidity and subcultured by trypsinization followed by splitting the cell suspension into new flasks and supplementing with fresh cell growth medium. Three days before the start of the experiment, the growth medium of near-confluent cells was replaced with fresh phenol-free DMEM, supplemented with 10% charcoal-dextran stripped FBS (CD-FBS) and 1% penicillin-streptomycin .
The experimental groups consisted of group 1 (G-I) with serumfree DMEM defined as the untreated DMEM. Group 2 (G-II) consisted of positive control (sildenafil citrate) at different concentrations. Further, group 3 (G-III) included DMEM medium (test item group) with the one-time Biofield Energy Treatment and denoted as BT-I, while the group 4 (G-IV) included the test item with the two-times Biofield Energy Treatment and indicated as the BT-II.
Biofield Energy Healing Treatment Strategies
The test item, DMEM was divided into three parts. One part of the test item was treated with the one-time Biofield Energy Healing Treatment by a renowned Biofield Energy Healer (The Trivedi Effect®) and coded as the Biofield Energy Treated DMEM (BT-I), while the second part was received the two-times Biofield Energy Healing Treatment and denoted as the BT-II. Further, the third part did not receive any treatment and defined as the untreated DMEM group. This Biofield Energy Healing Treatment was provided by a renowned Biofield Energy Healer, Mahendra Kumar Trivedi, remotely for
3 minutes. The Biofield Energy Healer was located in the USA, while the test item was located in the research laboratory of Dabur Research Foundation, New Delhi, India. This Biofield Energy Treatment was administered for
3 minutes through the Healer’s unique Energy Transmission process remotely to the test items under the standard laboratory conditions. Mahendra Kumar Trivedi never visited the laboratory in person, nor had any contact with the test item (DMEM medium). Further, the untreated DMEM group was treated with a “sham” healer for comparative purposes. The “sham” healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated samples were kept in similar sealed conditions for experimental study.
Assessment of PDE-5 Enzyme Inhibition
The cells were counted using an hemocytometer and were seeded at a density of 0.4 X 106 cells/well in DMEM with 10 % FBS in 6-well plates. The details test procedure was followed as per Branton et al. 2018 [38-40]. Increase in cGMP level was determined as the following equation (1)
% Increase in intracellular cGMP level = <(B-A)/A>x 100----- -- (1)
Where, B = OD of cells treated with test item and A is the OD of untreated wells (media treated).
Values were expressed as Mean ± SEM of three independent experiments. For multiple group comparison, one-way analysis of variance (ANOVA) was used followed by post-hoc analysis by Dunnett’s test. Statistically significant values were set at the level of p≤0.05.
High Glucose vs Low Glucose DMEM for Cell Culture - Biology
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