Science topic

# Sodium Bicarbonate - Science topic

A white, crystalline powder that is commonly used as a pH buffering agent, an electrolyte replenisher, systemic alkalizer and in topical cleansing solutions.
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What is the maximum amount of sodium bicarbonate that can be administered to a cow (e.g 450Kg weight) suffering from acute rumen lactic acidosis?
The amount of sodium bicarbonate is usually depending upon the degree of base deficit according to the following equation by Constable et al. (2017)*:
Total Base Deficit (mmol/L) TBD = BW (kg) + 0.6 + BD (mmol/L), where 0.6 is a factor reflecting the amount of bicarbonate in the extracellular space, BE is the number of mmol of bicarbonate to correct the acidosis and usually obtained by blood gas analysis of blood samples using blood gas analyzer.
Let's assume that the reading of blood gas analyzer gives BD as 15mmol/L
Then TBD = 450 + 0.6 + 15 = 4050 mmol/L. however, one gram of sodium bicarbonate gives 12 mmol bicarb., so the total amount of soidum bicarb for a 450kg cow with severe lactic acidosis and has a base deficit of 15 mmol/L would be: 4050/12 = 337.5g.
* Constable P, Hinchcliff K, Done S, Gruenberg W. Veterinary medicine: a
textbook of the diseases of cattle, horses, sheep, pigs, and goats. 11th ed.
Missouri: Elsevier; 2017.
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I am looking for an alternate method to measure percentage of carbonate impurity from sodium bicarbonate. The specification is <0.23% carbonate. There is a titration method using barium chloride to precipitate the carbonate and back titration for the bicarbonate. This will measure total carbonate/ bicarbonate and also bicarbonate only, in order to calculate the carbonate. I am not sure if this will be sensitive enough for small amount of carbonate, which is less than 0.23%. I try find other ideas or separation with different techniques, for example using ion chromatography, but it seemed that carbonate and bicarbonate not separable by IC by many articles. Any comment and suggested techniques or method for this quantitation. This was originally a project to find alternate method from the USP method using the carbonate apparatus (for sodium bicarbonate) since we have difficulty with the method.
This is now an old question. What did you do? How satisfactory was it? Vogel's Quantitative Inorganic analysis also has a titration method
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Hello, I',m having quite some difficulty growing Ramos (EBV-negative Burkitt lymphoma) cells. I'm using a modified RPMI 1640 culture medium to match ATCC's recommendations (RPMI 1640 + 10% FBS + 25 mM HEPES + 2 mM L-glutamine + 1 mM sodium pyruvate + 4500 mg/L glucose + 1,500 mg/L sodium bicarbonate). It has been over a week since I resucitated a cryovial, and although the cells are not dying, they are not multiplying either as expected. I have changed medium every 2-3 days as suggested by ATCC.
Any suggestions on what to do to make them grow?
Thank you!
Hi Adrián, I know it has been a while since you posted this question. I am facing the exact same problem, did you find a way to fix it and grow your cells successfully?
Best,
Zoé
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I have ordered UM-UC-3 cells and am wondering if the media I currently have is appropriate to use?
From the suppliers website, it states to use: EMEM EBSS + 2mM Glutamine + 0.1mM Non Essential Amino Acids (NEAA) + 10% Foetal Bovine Serum (FBS) + 1.5g/L sodium bicarbonate + 1.0mM sodium pyruvate.
We currently have MEM α, GlutaMAX with no nucleosides (https://www.thermofisher.com/order/catalog/product/32561037). This media has Glutamine, NEAA, sodium bicarbonate and sodium pyruvate already in it.
However, it was brought to my attention that this media mightn't be suitable to grow UM-UC-3 cells in and that I should instead use Minimum Essential Medium Eagle (https://www.sigmaaldrich.com/IE/en/product/sigma/m0325).
I was under the assumption that MEM α was just a modified EMEM that contains all the necessary extra supplements in it.
Is there any significant differences between the two?
Hi,
Best wishes..
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I am using TC-100 media(with serum and antibiotics) having sodium bicarbonate and L-Glutamine to grow Sf21 cells, but after I thaw them, they initially attach and grow for some time, then they fail to attach after 2-3 passages and eventually die.
What could be the possible reason for it? How can this be tackled?
I will like to suggest this manual page 16-17. It should assist you.
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Sample tube: crude enzyme+ 1 % casein (100mM sodium bicarbonate buffer pH-9) than incubate for 30 minute add TCA (5% in distil water) than centrifuge than take readings at 600 nm.
Blank1: water + casein, same as above
Blank 2: water + enzyme, same as above.
take readings from spectro. (Run Sample and Blank simultaneously)
It therefore measures the total reducing capacity of a sample, not just phenolic compounds. This reagent is part of the Lowry protein assay, and will also react with some nitrogen-containing compounds such as hydroxylamine and guanidine.
To determine the activity in a solid protease sample diluted in enzyme diluent, divide activity in units/mL by the concentration of solid used in this assay originally in mg/mL, which gives activity in terms of units/mg.
The phenolic compounds react with the Folin reagent which is formed by sodium tungstate and sodium molybdate in acid medium. Thus, this reaction produces a yellow complex that can be reduced with the phenolic compounds and identified with a spectrophotometric method at a wavelength of 765 nm (Cueva et al., 2010).
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I am working with MCF 7 cell line. I ordered the cell line from NCCS Pune. I received a very high confluent flask. After a day, I splitted the cells. I am using MEM w/Earle's salts, 2mM L-Glutamine, 1mM Sodium pyruvate, NEAA and 1.5 gms per litre Sodium bicarbonate with 10% FBS and antibiotics. A day after the splitting, I observed that all the cells were detached and floating. I changed the media and the flask again but next day the cells were in same condition ? They are not adhering in the flask and a lot of cells are dead. What should I do ?
When cells are approximately 80% confluent, they should still be in the log phase of growth and will require sub-culturing to ensure proper growth and health of the cells. When cells become over confluent, as you mentioned that they were too confluent, they will begin to die off and may not recover.
Did you check the cell viability percent during sub culturing? Subculture the cells in smaller culture flask (T-25) and give them some time to adhere. Usually MCF-7 cells adhere to the substratum after 8-10 hours. Otherwise, you may have to contact NCCS and request them for another flask if MCF-7 cells do not recover.
Best.
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Hi there,
I've been noticing that after adding my blocking chemicals (TEA-Cl and CdCl2) to isolate sodium current, my pH increases beyond my ideal point (I'm trying to stick around 7.4 and it drifts to around 8.0 after adding both) and there is first cloudyness and then precipitate forming in the solution after addition of the CdCl2.
Prior to adding the blockers, I am bubbling my ACSF with carbogen for twenty minutes. I also do not add sodium bicarbonate or dextrose to my 10X stock. My concentrations for ACSF (working solution) are as follows:
125mM NaCl
2.5mM KCl
1.25mM NaH2PO4
1mM MgCl2
25mM NaHCO3
25mM dextrose
2mM CaCl2
75mM TEA-Cl
0.2mM CdCl2
Again, target pH is 7.4. Would really like some input on this, as well any any relevant chemistry as to what is happening. Thank you!
Cadmium binds to phosphate and forms an insoluble compled that precipitates out of solution. If you wish to block calcium currents, leave out the NaH2PO4.
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Hi
I have Biosra brand DMEM high glucose powder medium, which I prepare according to the protocol. But after adding 3.7 g/L of sodium bicarbonate. I set the pH of the environment between 7.3 and 7.4. At first, the color and pH of the environment are good, but after a short time, the color becomes pink and the pH is alkaline. what should I do? Do I need to add HEPES? And if so, how much?
Dear Maryam, thanks for sharing your observations with other RG members. For useful protocols for preparing this an other culture media please have a look at the following links:
Media Preparation from Powder and Concentrates
and
Instructions for preparation of powdered medium
Also please have a look at the answers given to the following related question which has been answerd earlier on RG:
Good luck with your experiments and best wishes!
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I’m working on HIT_T15 cells , and I would ask about MTT assay!!
i Know that the medium should be without phenol red , but most of the mediums I found it available was without phanol red and l_glutamine or sodium bicarbonate also or without HEPES , what should i do in this case ? May any one help me please
you buy the media without phanol red and l_glutamine or sodium bicarbonate/HEPES. Then add the needed L Glutamine . sodium bicarbonate/HEPES separately.
L glutamine, Sodium carbonate and HEPES are available in packs separately. check the composition and add that quantity while preparing.
so your phenol rel is now not there and both sodium bicarbonate and HEPES are for stabilizing the pH, L Glutamine is a amino acid. it should solve your immediate need.
i was thinking of using a amino acid kit to make the whole medial with vitamins and glucose minerals will be a complex exercise. but people working with radioisotopes have to substitute a radiolable molecule they i think do so. The point i am trying to highlight is one can replace any component nor needed by preparing the whole media by getting all the needed ingredients.
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I’m working on HIT_T15 cells , and I would ask about MTT assay!!
i Know that the medium should be without phenol red , but most of the mediums I found it available was without phanol red and l_glutamine or sodium bicarbonate also or without HEPES , what should i do in this case ? May any one help me please
You need not go for another culture medium. Use the same medium which you have been using ( with phenol red) for MTT assay. Though phenol red and the final formazan product absorption spectra overlap, and phenol red can interfere with the formazan absorbance reading, there is a way out.
In your case you will have to use acidified isopropanol to solubilize formazan crystals. The acidified isopropanol will convert the phenol red in culture medium to yellow-colored form that will not cause any interference while recording the absorbance of the final solubilized formazan product. So go ahead and perform the MTT assay.
I hope I have helped you.
All The Best!
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Hi I am growing marine algae however i have lots of contaminants in my samples such as rotifers . I have attempted to remove these with sodium bicarbonate however the eggs still do remain. i have tried filtration with a 0.45um filter however the filter cloggs. Any suggestions on how i can remove these without losing the algae as the aim is to have a pure culture?
Thank you all .@md.Hamidur Rahman I am currently undertaking my masters project based at ushaka seaworld if I am successful then i would look at commercial.
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I found that there are two pKa values of sodium bicarbonate i.e 6.4 and 10.3. So can I take sodium bicarbonate to prepare buffer solutions of pH values around 6.4 and 10.3? Please suggest me.
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Is 0.2M sodium bicarbonate always has pH 8.3? I mean do you need to add HCl/NaOH to adjust the pH when you mix 0.2M sodium bicarbonate with water? Really confused about it.
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I know all the calculations so, the fundamental problem is that the solution is, I think, saturated and I can't dissolve 2 moles of sodium bicarbonate. Any suggestions?
A solution of sodium bicarbonate (NaHCO3) 2M means that its formal concentration is CNaHCO3 = 2M. Even if that may exceed the reported solubility at the intended temperature, such a solution can in principle be obtained in practice from a concentrate (possibly saturated) solution by first alkalizing the solution with NaOH, so that CNaHCO3 + CNaOH = 2M; and hereafter, carbonating the solution by sparging CO2 until complete conversion of NaOH to NaHCO3 by the dissolved CO2 (carbonic acid) can be achieved. After that, the solution can be kept stable by keeping the equivalent equilibrium partial pressure of CO2 in either the atmosphere above the solution (in principle a closed atmosphere), or in the sparged gas (for open atmosphere). I would say that by '2M NaHCO3 pH 10.0 sol.' we possibly can understand a mixed saturated NaHCO3 sol., with added NaOH up to a 2M sodium molarity (2M 'as NaHCO3'), hereafter adjusted to pH 10.0 by sparging CO2 into the solution. Such a solution would be then supersaturated if not stabilized as before mentioned. Of course; solution concentrations should be unambiguously reported.
According to the signalled link, the pH of sodium bicarbonate (saturated solution) should be found within 8-9; while 8.3 (at 77°F) was reported for freshly prepared 0.1 molar aqueous solution: https://pubchem.ncbi.nlm.nih.gov/compound/Sodium-bicarbonate#section=Experimental-Properties
You may want to check also:
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Soda bicarb is considered an ideal buffering agent to maintain rumen pH. Rumen has a large volume. Different literatures give different answers. How can we arrive at an ideal oral dose rate, interval and period for this buffering action in a practically possible manner?
What a great question.
I am still looking at it. However, I would like to described a little experiment.
Take two glasses one with carbonated water and another one with beer. Similar volume.
The differences between solutions explain CO2 holdup in the rumen. On the carbonated water you will see that bubbles are big and the quickly leave the glass. On the beer glass the bubbles are smaller and leave the solution very slowly.
The beer is like the rumen fluid. CO2 holdup: the dCO2 cannot easily leave the solution due to the physicochemical properties of the more viscous fluid. This effect limits bubbles formation and release (effervescence).
Now add to both solution sodium bicarbonate. you will see that the gas escape very quickly from both of them. In the carbonated water fizzles. But in the Beer it will form foam. This is the mechanism on Bloat and Abomasal dysplasia in cattle. You have destabilized the solution and CO2 will quickly be released leading to tympanism.
Do the same this time with NaCl and you will see the same effect as with sodium bicarbonate. This phenomenon call salting out the CO2 from the solution. And it is not an effect of bicarbonate, both solutions are saturated with HCO3-, but the Na which added to the liquid provides a substrate for nucleation (formation) of bubbles.
So, the answer is to add Na, the bicarbonate is the vehicle and a good one. Cl- might not be that desirable. how much? if your risk of acidosis is high add more to the diet. Here a good reference for a dosis/effect. (10.3168/jds.S0022-0302(97)76163-0)
I will also add that any compound that serves as a substrate for nucleation will have a good effect on preventing ruminal acidosis. Zeolites for instance are a good additive. But also, might be common sand.
Something that you might want to investigate.
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I have a body of water (volume=x) with about pH: 5.5 and alkalinity of about 4,5 mg/L CaCO3. I am looking to increase the pH to about 7,2 and increase the alkalinity as much as possible (no more than 200mg/L CaCO3). Is there a correct way to calculate the amount NaHCO3 needed for this? What other parameteres are needed for the calculation?
CO2: 5%
O2: 95%
i have used sodium carbonate to treat my water, but sincerely i forgot to calculate, what i did was i diluted few grams of the sodium carbonate in a bowl of water, after it dissolved i then introduced it into the pond containing only water, i stirred it well and allowed to dissolve, the next day i used my pH meter to ascertain the level of the acidity and alkalinity.
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Role of pH in RBC lysis solution
I want to ask aquestion in context of RBC lysis solution used in flow cytometry
We have been using our own lab made lysis solution containing ammonium chloride sodium bicarbonate and edta but when we keep the cells for more than 15-20mins in the solution out neutrophills convert into debris Or start to loose there integrity and shift to lower forward and side scatter.
Does pH play an important role here?
How long we can store the solution?
For RBC lysis step, I usually keep the cells (spleenocytes) in RBC lysis buffer for 10 minutes.
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Hi.
I want to perform a plaque assay using MA104 cell line. After virus absorption, I wash the cells with serum free media and add a 1:1 mixture of 1.2% agarose and Medium( DMEM containing L-Glutamine, Vitamins, Nonessential amino acids, sodium pyruvate, sodium bicarbonate and Pen/Strep). I put the plate in 37C and every 24 hours I check them to avoid gel drying. I check them every 24 hours but after 48 hours, cells die and I can not see any plaques.
Does anyone know how can I keep them alive to continue the test and observe the plaques?
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Recently Sigma and other companies are providing culture media that are modified for autoclaving. For example: Auto-Mod™, without L-glutamine and sodium bicarbonate, powder, suitable for cell culture (Sigma R7755). Do these autoclavable media work as well as sterile-filtered media?
Dear Afzal,
In the same situation, I have used a vitamin B12 assay medium was obtained from Biomark. It was autoclavable media and worked well when it inoculated with bacteria. The main properties stayed unchanged.
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Hi everyone, I am currently culturing NK-92 cells in the ATCC recommended culture medium (Alpha-MEM without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate. With: 0.2 mM inositol; 0.1 mM 2-mercaptoethanol; 0.02 mM folic acid; 100-200 U/ml recombinant IL-2; 12.5% horse serum and 12.5% fetal bovine serum)
The culturing has been successful before for a few weeks however now when using a thawed out sample (from liquid nitrogen) I see under the microscope that the cells are not forming the proper (grape-shaped) clumps. In fact they are quite far from globular shaped and are very oddly-shaped. Has anyone had this issue before? My coworker is culturing the same thawed-out cells in the same medium but hers form proper clumps, while mine are very strangely shaped. Any suggestions or ideas would be appreciated, thank you!
I agree with Malcolm Nobre but would seed them at a density of at least 0.5X10^6 cells/ml with freshly prepared media
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I´m currently working with the THP-1 cellline.
I have two questions.
1. I got those cells as a gift from a nother group which told me to cultere this cellline in RPMI + Glutamin + Pen/Strep.
i recently read that they also need 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate an beta-mercaptoethanol.
Are those compounds necessary?
2. Is there a protocol for neutrophil differentiation?
I found a protocol using dibutryl cAMP would this work ?
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I'm working with Herpesvirus and Adenovirus. For Herpesvirus, I use Vero cell line to activate and check virus titer. For Adenovirus, I use Hela cell line.
Recently, I could activate them, but can not have high titer. The titer i need around 10^9 or 10^10 TCID50. But I just have around 10^5 TCID50.
I use this protocol to activate virus:
1. Cell cultured in 75cm2 flask for 2- 4 days, until 90 - 10% confluent.
2. Wash 2x with PBS.
3. Infect 3ml of virus stock for each 75cm2 flask for 90mins, rock the flask every 15 mins to deposit all cells in flask.
4. After 90 mins incubation with 3 ml of virus stock. Add more 20 ml of medium (DMEM+2%FBS+1%P/S + 44mM Sodium bicarbonate).
5. Incubate flask for 2 - 3days until 90 - 100% CPE appears.
6. Freeze (-70 degree) and thaw (4 degree) 3x to lyse cell.
7. Cfg 1500xg, 4 degree, 10 ml, collect supernatant for virus stock.
Could you please show me specific protocol to increase these viral titer? Thank you so much.
Amanda Westergren Jakobsson . Thank you so much.
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I extracted phosphate from my sample using a sodium bicarbonate solution. When I acidify the sample to a very low pH, I see that the color development (using ammonium molybdate and ascorbic acid) takes longer than when I directly add the reagents to the extract without acidification. However, I understand that when I add the reagents without acidifying, carbon dioxide is produced due to the presence of bicarbonate.
In this case, would it be more ideal to adjust the sodium bicarbonate solution to around pH 6-8? I read somewhere that this is the ideal sample pH.
Kendric - thank you for this interesting question. Although I'm an inorganic chemist, I'm certainly not a specialist in this particular field. However, it might be helpful reading the article entitled "Stabilization of Molybdate Reactive Phosphorus in 0.5 M Sodium Bicarbonate Extracts of Soils" which is available as public full text on RG:
According to this article, there is a significant difference in the pH values during the extraction and the phosphate analysis. For the phosphate extraction, the pH of the sodium bicarbonate (NaHCO3) solution must be adjusted to 8.5. The subsequent colorimetric analysis must be done under acidic conditions. For this purpose, the exctract is acidified with sulfuric acid (H2SO4). Under these conditions the bicarbonate decomposes under evolution of CO2. This is OK because at this stage the bicarbonate is not needed any more.
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Sodium Bicarbonate intraperitoneal dose for adult patients using PD solution containing of Lactate. How often can be used? And what are the Precautions should be taken during the administration
Optimum bicarbonate concentration in peritoneal dialysate to reduce pain
The pain during peritoneal dialysis usually occurs early period at the onset of this mode of dialysis. This is usually due to acidic nature of the peritoneal dialysate fluid, either due to lactate or due to hypertonic dextrose or both. Usually optimizing the bicarbonate in the peritoneal dialysate fluid can reduce the pain.
However, be vary of increased incidence of peritonitis by use of high bicarbonate.
You can choose the ideal regimen of bicarbonate concentration in the peritoneal dialysate fluid as guided by the regimen described in the two articles (1-2).
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When I run a solution containing dissolved salts of Mg, Ca,, and Na and as well sodium bicarbonate through a Nylon based 1um filter, I noticed that pH increases.
I am concerned that some salts are removed. Do Nylon based filters do remove some ions or it could be only that H+ ions stick to te Nylon membrane?
Hey Ilija, I hope you are doing well these days.
Nylon has a tendency to absorb quite a bit of water. Roughly 10% by weight is not uncommon. If the volumes you are filtering are fairly small, then you are likely to see some effect on your solution as a result of the loss of water into the nylon.
You may want to switch to a PTFE filter.
Best regards,
Ron
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I want to culture Spirulina in a large scale. but for example a 10,000-L medium needs to 160Kg Sodium Bicarbonate according to Zarrouk!!! I have a protocol that requires 8 g/L of Sodium Bicarbonate, which is still too much. i need a protocol to reduce the volume and cost. would you please help me?
I have gathered a few recipes from artisanal spirulina producers, and it seems hard to go below 8 g/L of sodium bicarbonate. However, it is possible to run in fed-batch or continuous mode with diluted media : we once ran a 30-L batch inoculated at 1 g/L spirulina, in a Zarrouk medium diluted 5 times, and had quite good growth curves during 3 days without adding any nutrients.
You may replace sodium bicarbonate by some other source of alkalinity (sodium carbonate for example) if you have the possibility to inject CO2 in the reactor. But in my opinion, the best solution to reduce nutrient costs is to recycle the medium after harvesting.
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I want to culture Spirulina in a large scale. but for example a 10,000-L medium needs to 160Kg Sodium Bicarbonate according to Zarrouk!!!
I have a protocol that requires 8 g/L of Sodium Bicarbonate, which is still too much.
Hi Nasrin. you need to use the sodium bicarbonate just once and after harvest just add another nutrient except sodium bicarbonate. Also if you use 8 g/l of sodium bicarbonate and 8 g/l of sodium carbonate you will achieve better results.
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Method and solvent
You can add water sodium bicarbonate will dissolve in water, To this add ethyl acetate, The organic acids will dissolve in organic solvent. Separate the organic solvent and water using separating funnel.
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I'm formulating a natural deodorant and found that the 5% concentration of NaHCO3, it works great for inhibiting the scent of sweat, but about 15% of subjects get a rash with chronic usage, ie daily for weeks.
Would 1% still have an anti-microbial effect to prevent odor?
could be written on canes as ingredients?
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Hi, I am trying to dissolve pure reagent grade Quercetin into cell culture medium (DMEM+sodium bicarbonate+antibiotics) but it clearly does not form a solution. I've found that DMSO might serve but high DMSO concentrations are toxic for cells. Does anyone have found a non-cytotoxic method for dissolving quercetin? Thanks in advance.
Dissolve quercetin in 10mM DMSO in cell culture medium as a stock. Dilute the stock solution in cell culture medium usually up to 0.2% of DMSO is non-toxic to the cells. 0.2% of DMSO used as a negative control.
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I am going to be growing NK-92 Cells with the ATCC recommended media recipe below. I am trying to make frozen aliquots of all of the components for ease of making future bottles of media and the folic acid recommends dissolving in 1M NaOH. My other option I have seen is dissolving in DMSO, which I am not necessarily comfortable with growing cells in DMSO containing media long-term. Someone recommended on another post to just dissolve it straight into the media, however measuring out 4.4mg for 500 mL of media allows for much more room for weighing error than I would like.
Will using 1 M NaOH be fine for the cells provided I make the folic acid as concentrated as I can to add the least amount of NaOH possible? Or am I able to use something a little less harsh like sodium bicarbonate to dissolve the Folic Acid? Any other recommendations for cell culture help are also welcome.
NK-92 Media:
Alpha Minimum Essential Medium w/o ribonucleosides and deoxyribonucleosides
2 mM L-glutamine
1.5 g/L sodium bicarbonate
0.2 mM inositol
0.1 mM 2-mercaptoethanol
0.02 mM folic acid
100-200 U/ml recombinant IL-2
12.5% Horse Serum
12.5% Fetal Bovine Serum
Yes, you can make stock solution in NaoH and further diluted in incomplete medium.
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Hey there,
I'm using commercially available Drabkin's reagent to measure the hemoglobin content of blood photometrically. The reagent I bought contains sodium bicarbonate, potassium ferricyanide and potassium cyanide. In my understanding, all different forms of hemoglobin (except sulfhemoglobin) are converted to methhemoglobin by potassium ferricyanide in a first step which reacts then with potassium cyanide to cyanomethemoglobin which shows a significant absorbance at 540 nm.
But sometimes I see a second peak around 575 nm (indicated by the red arrow in the graphic), sometimes very weak but sometimes also very strong and I wonder what it could be. Could this be some remainings due to uncomplete reaction or could it maybe be due to poor quality of the sample?
Oxy-Hemoglobin typically has peaks at 541 and 576 nm. Because these peaks are still present in the spectra you showed, your sample likely still has some amount of unreacted hemoglobin which will impact your results. Typically this lack of complete reaction is caused by 2 factors:
1. Insufficient Drabkins reagent
2. Not enough time for the reaction to reach completion.
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I have noticed some studies report using sodium bicarbonate and some report using sodium carbonate to determine total phenolic content of their samples. My question is does it matter which one to use and what concentration as the range is anywhere from 7.5% to 20% solution. Thank you in advance for any suggestion.
Which method " to determine total phenolic content " you are asking about? It might be just a way to reach a desirable pH.
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I'm working with trace amounts (say, 1 microgram) of sterols such as dinosterol, stigmasterol, and cholesterol, and have been trying to acetylate with acetic anhydride and pyradine. This is a standard procedure our lab has performed for years. Transfer the sterol to a vial insert with toluene, evaporate, add 20 ul of anhydride and 20 ul of pyridine, heat to 70C for half an hour, cool, evaporate reagents. For several months now, though, the sterol acetate yield from this step has been 20-30%, instead of our typical 90-100.
The confusing part is that it appears that sterol is being destroyed/converted, not just failing to react. When I acetylate a sterol standard and get 30% yield of the acetate, I don't see un-derivitized sterol in my chromatogram. Furthermore, if I sylilate the residue with BSTFA/TMCS, I do NOT recover the un-acetylated sterol as a TMS ether, either. If I sylilate an un-reacted sterol standard, however, I get 100% of what I expect as the TMS ether (so I know it's not a problem with my standard being more dilute than it should be). This does NOT happen with n-alkyl alcohols. nC21-OH, for example, acetylates with 100% yield just fine.
I have tried new containers of acetic anhydride and pyridine. I have tried diluting the mix with toluene and/or changing the ratios of anhydride/base. I have tried changing glassware, or using teflon. I have tried purging the reaction vial with nitrogen and argon. I have tried substituting triethylamine for the pyridine. I have tried water/DCM phase separations followed by drying of Na2SO4 as a workup instead of simply evaporating the reaction mixture. I have tried pre-deactivating my glassware. I have tried letting the reaction proceed at room temperature overnight. I have tried leaving the reaction at 70C overnight. I have tried a different method that uses toluene, acetic anhydride, and sodium bicarbonate as a sparingly-soluble base that keeps the byproduct acetic acid quenched. I've compared results on a GC-FID with a PTV inlet, a GC-MS with a split/splitless, and someone elses GC-FID with split/splitless in another lab.
Nothing has worked.
Does anyone, in the name of all that is good, have any idea where my sterol might be going? Or how to proceed to fix this? Simple anhydride:pyridine at 1:1 was always effective in the past, and the lack of un-reacted sterol tells me it's not a reaction rate problem that a catalyst will solve.
Regardless, I currently have test reactions running that include DMAP and n-methylimidazole as catalysts. I haven't worked them up yet, but for former turned deep yellow and the latter turned deep red. The reactions in which I used TEA as the base also turned yellow, and left behind a large amount of oily residue. Are these warning signs or telling indicators of some sort of oxidative problem?
In a couple of days I will have materials for methods using silver triflate or montmorillanite clay as catalysts, but I have no reason to suspect these will go any differently.
Edit: Edited to indicate that, in contrast, straight chain saturated alcohols DON'T seem to have any problem acetylating with 100% yield under normal conditions.
In this case it's because we're derivitizing for isotope analysis. We need the protecting group to be A) non labile and B) have the minimum number of hydrogens and/or carbons on it. TMS is great for quantification but doesn't work well for compound-specific IRMS.
Thank you, I will try those folks.
Any ideas on the yellow/red reactions?
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I would like to know what else need to be added to make complete medium that can be used in 5% CO2 incubator, do we have to add Non Essential Animo Acids and Sodium Pyruvate, and HEPES apart from sodium bicarbonate? and how much amount Sodium bicarbonate needs to be added to prepare 1L medium to be used in 5% CO2 incubator. To be more specific, Gibco Minimum Essential Medium powder, catalog number 61100-061
Muruganandan Shanmugam , Thank you so much Sir for your response, I would also like to know about Non-essential Amino Acids and Sodium Pyruvate. Will be waiting for your reply, exact product that we purchased is Gibco Minimum Essential Medium powder, catalog number 61100-061, description in the below link
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I’m having trouble in culturing THP-1 for over 2 months now. I followed the protocol you provided in your web page. We are establishing this cell line in our lab so we got a frozen vial from different research center located in different city and the vial was transferred in dry ice. When I got the vial it says that viability is 99%. And I couldn’t get any information about how that vial was cryopreserved since the center are not working in this cell line anymore and the vial was stored at Liquid nitrogen since 2005. Mean wile I thawed the vial using RPMI Medium 1640 from Gibco with (+ 4.5 g/L D-Glucose + 2.383 g/L HEPES Buffer + L-Glutamine + 1.5 g/L Sodium Bicarbonate + 110 mg/L Sodium Pyruvate) and followed the steps below:
Day 1(29% viability):
1. I prepared complete media + 20% FCS + Glutamine in case was degraded in our media.
2. I thawed the vial for 2 minutes
3. I decided to leave it settle down for 2.5 hours in the incubator
4. I centrifuge for 5 minutes at 300g
5. I discard supernatant and resuspend pellet in 5 ml of media
6. Transfer cell content into new 25 ml flask and keep upright in the incubator and see after 48 hours
7. From beginning I noticed that the live cells number is very small compared to dead ones.
8. Viability was 29%. HOWEVER, Viability of the vial was 99% and method and condition of cryopreserving of that vial was unknown.
Day 2(27% viability) 3days (from thawing):
1. I prepared complete media + Glutamine
2. I decided not to do any centrifugation , just add new 10 ml complete media+ Glutamine
3. Leave flask in incubator over weekend
Day3 (6% viability) 6days (from thawing):
1. I notice that number of live cells has dropped and viability is 6%
2. I notice that media is turbid and very small thing appear under microscope ( may be Dead Cells Debris)
3. I centrifuge for 5 minutes at 300g
4. I discard supernatant and resuspend pellet in 5 ml of media ( 2 ml of old medium + 3ml of new medium)
5. Transfer cell content into new 25 ml flask and keep upright in the incubator and see after 24 hours
Today … 7 days (from thawing):
1. I centrifuge for 5 minutes at 300g
2. I discard supernatant and resuspend pellet in 5 ml of new media (no old media)
3. Transfer cell content into 6 well culture plate and keep in the incubator and I will see after 24 hours
I would appreciate the chance to have your advice in my steps and how my culture of Cell look like.
I have attached my cell culture for all 4 days.
Your main mistake is made in the beginning when you thaw the cells.
To preserve cells and avoid intracellular ice formation the freezing medium is enhanced with glycerol etc. to pull out the water from intracellular space. Therefore when you leave the cells after thawing for 2.5 h in freezing solution without washing it out you are simply intoxifying the cells.
Therefore to preserve the cells viability you have to change your protocol. Before you launch thawing at 37C prepare in advance the medium with 10%FCS/FBS and and 1% of antibiotics (50.5 units/ml penicillin, 50.5 μg/ml streptomycin and 101 μg/ml neomycin, Sigma). Fill the 15mL falcon with 10mL of your full medium (FBS+ antibiotics) and add the thawed cells suspension. Mix it delicately in hands and immediately centrifuge to remove all toxic agents. Remove the supernatant without disturbing the cells pellet. Then re-suspend the cells pellet in full medium and seed the cells in TC flask.
Usually after two days cells should be already well attached and you should change the medium. Then after leave the cells for about three days - observe the medium color change as an indicator of a pH. Add fresh media and wait till the required cells number will be achieved and then divide them into new TC flasks.
Good luck!
Katarzyna
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On two visits to Tenerife my long term digestive problems stop within a day. On investigating, I find that adding Silicon 30mg /litre and sodium bicarbonate to bring the total alkalinity to 240 to match the water in Tenerife has the same effect. Why ?
I have investigated all the other parameters you mention and although they may have minor effects. I have managed to obtain the major factors by reproducing them in water in the UK with the addition of Silicon dioxide ions (potassium silicate) and Sodium Bicarbonate to increase the total alkalinity. This water now has a similar mineral content to bottled water from Teneriffe.
What I wanted to know is more about the biochemical processes involved in the use of Silicon in digestive mechanisms. I know Silicon is important for connective tissue and is used to improve hair and nails but what other functions does this mineral have.
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It was observed that the drug released from microspheres decreased as the concentration of sodium bicarbonate increased
The second factor is the drug polymer ratio of the same polymer, increasing the polymer amount will cause more retardation of the release so the optimum ratio of the drug to polymer is required for selected release
A third factor is the porosity of the formed particle and the presence of surface pores could facilitate the release
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1-During anaerobic treatment of wastewater, sodium bicarbonate is added for alkalinity and pH control. I already know how to calculate this sodium bicarbonate dose.
2-If a energy mass balance is done around this anaerobic treatment, energy is produced in form of methane, but how could I calculate the energy entering the system in the form of sodium bicarbonate?
Thanks for your answer. I´ll see what I can find in the literature.
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I used Gibco RPMI 1640 ( 4.5g/L D-Glucose, 2.383 g/L Hepes Buffer, L-Glutamine, 1.5g/L Sodium Bicarbonate, 110mg/L Sodium Pyruvate) medium supplemented with 2% Human serum for monocytes plastic adherence and then i used for macrophages culture the same medium composition plus 50ng/ml GM-CSF to generate M1 macrophages and 50 ng/ml M-CSFfor M2 macrophages.
The problem i have , is that i don't get enough macrophages after 5 or 7 days differenciation and the M1 macrophages are CD206 positve and low CD 86 positive whereas the M2 macrophages are CD206 positive and CD86 positve.
If someone has a good Media composition please share with me
Agree with Giulia about the media and don't forget to activate them with LPS/IFNg (M1) and IL-4/IL-13 (M2). About the markers , CD209 works quite well for M2 and CD80/CD86 for M2.
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After some consulting I decided to use a commercial pcr cleanup spin column kit to concentrate DNA from my CHIP for sequencing. The DNA was in elutate of sodium bicarbonate and 1% SDS. Instructions required me to dilute 6x in binding buffer, so this meant 9mL per sample column. When combined, precipitate came out, I assume this was the SDS. All of the protocols I've seen that use sodium biocarbonate and SDS to elute out dna jumped straight to purification using p/c or kits so I just moved on.
I ran the liquid through the column and near the end when I was washing them I noticed that the membrane of the tubes looked a bit crumpled and the wash buffer was dripping through it somewhat faster than in control columns and whitish precipitate was in the wash. I tried looking at the membrane at every angle but I didn't see any hole. After I eluted I noticed a tiny pellet of whitish stuff at the bottom of the tube. I'm not sure whether this was SDS or silica or what. I had the elutant tested through qubit and the samples showed very low to below threshold amounts of DNA. But this was CHIP pulldowns so this is not necessarily indicative that something is wrong. I was wondering what sort of steps I should take from here. Should just move on and see if I can create a library and do a PCR check or assume the columns were broken and try to reprocess the binding buffer waste which I kept? Multiple companies I contacted seemed to think that the spin columns should be able to withstand that volume although one of them (Thermofisher) apparently has columns with membranes that look thicker than the kit I was using.
How much time, effort and money have you spent up to this point versus how much you will spend if you proceed with what you have?
If the CHiP part is easy enough to repeat, I would do that rather than proceeding with dubious material.
Also I would avoid bicarbonate, unless there is a very good reason that CHip will not work without it! Also 1% SDS seems to be a huge concentration!
I suspect that the membrane has been shrivelled up by the buffer components, but you might simply try exposing a purification tube to buffers alone to see what happens.
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Can we use DMEM medium for hybridoma cell culture adding Sodium bicarbonate without keep in a CO2 incubator? Because at the moment we are using MEM medium without CO2 incubator for cancer cells. Therefore, I need to clarify whether we can use DMEM in same CO2 free incubator for hybridoma cells.
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I am developing whey protein beverage that sterile by retort. I have to adjust pH of product from 6.5 to above 7 to prevent protein coagulation during retort process.
I think I will use sodium bicarbonate to adjust pH. Could you give me suggestion? If you have another idea please let me know.
Regards,
Hi,
You can use buffer solution . The buffer is best than sodium bicarbonate
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We are using above mixture for our cancer cell culture. I need to know whether we can use the same medium for hybridomas, instead of RPMI or DMEM which is mentioned in the product details. pls reply me.
Can we use DMEM medium for hybridoma cell culture adding Sodium bicarbonate without keep in a CO2 incubator?
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I'm running a conjugation reaction to label 20nt oligonucleotides with an NHS ester fluorophore. The oligos have a 3' amino group to anchor the ester.
I dissolved the NHS ester in 100% DMSO and the oligos in 0.1M sodium bicarbonate (pH 8.4), and added them together into a single tube for overnight reacting at 4˚C.
As soon as I added the NHS ester to the oligos, I noticed a bunch of small solid particles precipitate. Is this the DNA? Will this likely result in the reaction not proceeding or getting very little conjugated oligos? Any recommendations on dissolving this precipitate?
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As we know, the chemical pesticides or fungicides in the fruit are not eliminated by washing. Since these toxins are soluble in water, does the immersion of fruits in water (from 20 min for grapes to 8 h for citrus) or sodium bicarbonate (baking soda) 1%, eliminates these toxins from the fruit?
I'm not agree. It's suggested use the different agricultural poisons in optimum range. I think the world work on decrease using poisons Technic and methods. We know that the outer layers of fruit are covered with a wax that is resistant to water penetrate.
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Hello everyone,
i work on various cell lines, recently i procured THP-1 cell line, which i am unable to culture in regular DMEM media with high glucose without sodium pyruvate and low sodium bicarbonate along with 50um of 2-mercapthoethanol, can anyone suggest what will be suitable culturing media for THP-1 cell line and any other components to be added for their culture.
Thank you.
We use advanced RPMI (Gibco), supplemented with glutamax, 10% heat-inactivated FBS, plus the highly documented beta-mercaptoethanol (50 uM). Seeding density is about 10^5 cells/mL and should never exceed 10^6 cells/mL. You need to gently blow the cells routinely to disperse them while doing maintenance or passaging. Observe frequently to prevent lumps formation. Good luck
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I plate MDCK cells in 12-well plate and make sure next day wells are a confluent monolayer. Then I aspirate the cell culture supernatant and wash with PBS twice, and add 200 ul of tenfold virus dilutions to each well and incubate for an hour in 37 at RT for 1h and rock the plate side to side every 10 min. Then I mix and add the agar overlay which consists of sterile water, 2x MEM, 1% DEAE dextran, 5% sodium bicarbonate, 1 ug/ml trypsin-TPCK, and 2% Oxoid agar. The agar solidifies at room temperature for 15 minutes before placing in the incubator. After 24 hours, the cells are all dead. So I have tried a lot conditions (to make sure the agar was not too hot and to change ingredients of agar overlay). Finally, I found all MDCK cells without trypsin-TPCK in agar overlay survived. Does someone can tell me what happened to my trypsin-TPCK (they were prepared in 2016 and stored in -20). And Now I have ordered new trypsin-TPCK, the instruction showed it should be dissolved in 1mM HCl (ph=3), while someone said dissolved in DMEM or sterile water, I'm not sure about it. any suggestion?
I did my PhD in a flu lab and remember we often had issues with plaque assays not working! We never fully worked it out but here is some anecdotal advice.
As a gral suggestion we felt like using a relatively 'fresh' batch of MDCKs was important (not too many passages before the assay, ideally only 3-6 splits and no more than 10).
Specifically in your case, maybe the way you are adding the trypsin, directly to the cells, is killing your cells?
One difference to your protocol is that we added the trypsin to the overlay medium. If I remember correctly, the incubation with virus is for these to stick to the cells via HA-sialic acid. Then the overlay medium provides the trypsin needed for HA cleavage and infectivity - but don't quote me on that... it's been a while!
We never suspected about the trypsin and we got ours from Worthington (ScimaR), 10mg dissolved in 100mL PBS and then 0.45um filtered in a sterile hood (store at -80). We added 200uL of Trypsin to a 100mL of overlay medium.
I hope this helps. Good luck!!!!
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Does anyone has prepared RNC(Rany Nickel catalyst) of 85-90% Purity?
We using leaching method with NiAl alloy treated with Sodium Bicarbonate and it gives only 15% purity.
What promoters,dopping should enhance the ratio?
Kindly share your thoughts, experience ASAP.
:)
I should have written "Raney Nickel" but it's my cellphones not-so-smart dictionary that cause the error.
I was in kind of hurry so .... Nevermind
Thanks for the correction.
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Is it necessary to boil dialysis tubes in EDTA/sodium bicarbonate before using? or is it enough to boil it in distilled water? What is the recommended protocol for preparing dialysis tubing?
These are the instructions given by Sigma for preparing their dry cellulose acetate dialysis tubing.
"Removal of glycerol included as a humectant can be accomplished by washing the tubing in running water for 3–4 hours. The manufacturing process results in the presence of residual sulfate salts in the ppm range. Removal of the sulfate salts can be accomplished by treating the tubing with a 0.3% (w/v) solution of sodium sulfide at 80 °C for 1 minute. Wash with hot water (60 °C) for 2 minutes, followed by acidification with a 0.2% (v/v) solution of sulfuric acid, then rinse with hot water to remove the acid. This tubing will retain most proteins of molecular weight 12,000 or greater."
Spectrum recommends the following procedures:
"Preparation instructions will vary based on the membrane type as follows. Note that boiling membrane is not recommended as it can damage the membrane and alter the pore rating.
A) Biotech RC, CE, and PVDF membranes should be rinsed in DI water for 15 to 30 minutes to remove sodium azide preservative.
B) Spectra/Por® 7 Standard RC has been pretreated to remove the trace levels of heavy metals and sulfides and only requires a 15 to 30 minute soak in DI water to remove the sodium azide preservative.
C) Spectra/Por 1 through 6 Standard RC membranes may require some extra preparation. While rinsing Spectra/Por 1 through 6 in water is typically sufficient to remove glycerin or preservative, Spectrum offers two membrane pre-treatment solution kits for the removal of the trace levels of heavy metals and sulfides introduced during manufacturing. Heavy Metal Cleaning Solution and Sulfide Removal Solution Kits are recommended for ultra-sensitive dialysis applications like binding studies or when low level presence of these contaminants may interfere with downstream analysis of the dialysis sample."
The method of preparation of the tubing varies with the supplier and the type of tubing. Check with the supplier of your tubing. If your application is very sensitive to the presence of metal ions (e.g. DNA), treatment of the tubing with EDTA would be a good idea to bind up any contaminating metal ions. Additionally, the EDTA can be used as a preservative for storing the tubing.
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Could anyone help me to explain why my mouse embryonic cells will begin die when I add some reagents into the cell medium to differentiate ESs? Mouse ES grow on MEF. Cells will be exposed in MCDB 131 medium supplemented with 1.5 g/l sodium bicarbonate, 1×Glutamax, 10 mM final glucose concentration, 0.5% BSA, 100 ng/ ml GDF8), and 3 mM of CHIR-99021 on day 1, day 2 and days almost the same reagents. I will add some new reagents like 0.25 mM ascorbic acid and 50 ng/ml of FGF7 on day 4 and day 5. Day 6- and 7 need to add new reagents like 0.25 mM SANT-1, 1 mM retinoic acid, 100 nM LDN193189, 1:200 ITS-X , and 200 nM TPB. However, my cell will always die from day 1. Sometimes my all ES cells die after day7. Why, please help me and give me some advice.
thank you for your adice, i need to adjust to my protocol to let most of cells alive in the plate,
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What is the mechanism that reverses this condition ?
When administered, sodium bicarbonate dissociates into a molecule of sodium and bicarbonate. The molecule of bicarbonate in turn binds hydrogen converting into carbonic acid with its subsequent dissociation into carbon dioxide and water.
The benefit of sodium bicarbonate in the setting of TCA overdose is probably due to both an increase in serum pH and the increase in extracellular sodium.
Alkalization favors the neutral form of the drug and reducing the amount of active cyclic antidepressants. The high sodium load increases the electrochemical gradient across cardiac cell membranes, potentially attenuating the TCA-induced blockade of sodium channels.
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I am preparing a modified lectin based immuno assay. I am oxidizing my monoclonal anti-PSA antibodies (Abs) to avoid non-specific lectin attachment to the carbohydrated present of the Abs. I am following the following approach for oxidation.
1) Removal of sodium azide from Abs using desalting (spin filteration)
2) Reconstitution of Abs in PBS buffer.
3) Addition of sodium periodate solution for oxidation (20mM sodium iodate, 100mM sodium acetate, 150mM of NaCl - solution made in DI-water). Add equal amount of the solution to the Ab solution, at 4C in dark and keep it for 2 hours.
4) Removal of oxidized antibodies and reconstitution in sodium bicarbonate buffer (pH9.6). This avoids self conjugation of the Abs with themselves from the amine side.
5) Conjugation of NHS-PEG-PC-biotin linker using the generic biotinylation protocol.
6) Removal of excess biotin and blocking of excess aldehyde sites with 1M ethanolamine solution.
7) Removal of excess solution and extracting biotinylated Abs.
I want to block the oxidized Abs and want to know whether my approach will be fine ? I want to conjugate the biotin linker from the amine side using the NHS chemistry. I have seen oxidized Ab blocking with MPBH + Cys-Gly dipeptides. I am not sure whether this blocking mechanism will affect my biotinylation ?
4) Schiff bases will be potentially produced once you oxidized antibody. High pH favors this reaction.
You can use ethanolamine to block aldehyde groups.
If MPBH is:
(4-(4-N-maleimidophenyl)butyric acid hydrazide)
then they are using it to attach the dipeptide to the oxidize carbohydrate.
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While managing Patient with hyperkalemia ,we administer sodium bicarbonate !how does it work , or mechanism in action ?
Administration of sodium bicarbonate increase the concentration of Na+ in the extracellular fluid compartment. High extracellular Na+ concentration leads to its increased cellular uptake via the sodium-proton exchanger. The subsequent movement of Na+ out of the cell creates an electric gradient, forcing K+ into the cell via Na+-K+-ATPase, resulting in net increase in intracellular K+ .
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For alkaline soils, Olsen's method using 1M sodium bicarbonate is recommended for determination of soil available P.
However my ICP-OES technician says that because there is Na concentration in extract, P cannot be determined.
Known solutions:
1) Use UV-visible spectrophotometer for P estimation from the extract.
2) Use Colwell P method.
3) remove Na in the soil extracts and then determine P in the extracts.
Requesting experts to suggest the best solution. I have access to ICP at convenience. UV-visible spectrophotometer is not available currently.
Kindly help me in this matter.
Merry Christmas to all!
Thanking you,
Best Regards,
Rohan
Dear sir, thank you for the suggestion. I am looking for extraction procedure for available P in soil.
Acid digestion will not give desired results. I will once again read the attachments to see if I have missed out on suitable extraction procedure.
With Best Regards,
Rohan
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Hi, I am in need of a good growth medium for HepG2 cells. i know there are a lot of people using different types with varying additives and glucose but does anyone have experience using Williams’ Medium E liquid, sterile-filtered, suitable for cell culture, With sodium bicarbonate and L-glutamine, without phenol red ? I like to exclude the phenol red because it interferes with MTT protocols.
Hello Antonio,
in our lab, we are used to use DMEM or RPMI 1640 supplemented with FBS, Pen/Strep and Glut.
Naturally, you may find a lot of growth medium better or more specific than these, but I don't find any problems in my experience.
Good Luck!
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Due to the bacterial actions, the pH of the liquid media always stay acidic (about 4 - 5). For the purpose of maintaining the pH alkaline (above 8), dilute NaOH solution was initially used to adjust the pH on daily basis, then we thought about using the sodium bicarbonate as an alternatives.
However, after adding the baking soda power in the media, the gas production  ceased, the pH stayed quit stable (about 9 above). But I am wondering if the baking soda killed the bacteria or micro-organism ?
Is there anyway we can test if the bacteria is still alive?
Does anyone have the experience of keeping the pH alkaline of the bacterial media?
Thank you, Alireza
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Hi,
I recently bought DMEM/f12 without glutamine and sodium bicarbonate from Corning cellgro. Before using media I add glutamine, gentamicine and B27. This time media color change to orange-yellow. I checked PH and it is around 7.5. I am just wondering what is the reason of color change, is there any reaction between media and these supplements? I am not sure that I can use it or not.
Thank you dear Mariel. I put a little of media in incubator yesterday and I check that today it becomes a little close to red. I think it is fine to use it.
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Companies formulate DMEM always with 3.7g/L of sodium bicarbonate. But majority of researchers use only 5% CO2 incubators, which is ideal for media with 1.5-2.2g/L of sodium bicarbonate. 3.7g/L of NaHCO3 requires 10% CO2. So, while most of us use only 5% CO2 incubators and everyone knows this, companies must have a good reason for insisting on 3.7g/L. Do you have an idea on this? Does DMEM have to contain 3.7g/L NaHCO3 for some reason?
People in my lab also use DMEM but they didn't know DMEM requires 10% CO2. I let them know this but they are not willing to change their culture conditions as all of their experiments have been done in 5% CO2 all the time. So I decided to buy DMEM with lower NaHCO3 concentration. I found one from ATCC (1.5g/L), but they only have high glucose. 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. Do you have a suggestion about what to do? Should I just ignore these questions in my mind and keep doing what many researchers have been doing wrongly (i.e. using DMEM with 5% CO2)?
Sorry guys, I didn't check the thread for long time.
Adrian already shared a reference (I guess that is the only source where you can find the equation in this form) but they don't show how they get to that equation. Actually you don't need a reference, you just need to write down the chemical reactions and calculate the pH. Let me explain how you do that.
In bicarbonate buffering systems, the reactants and products are in equilibrium as shown below:
• CO2 + H2O <--> H2CO3 <--> HCO3- + H+
Let's first focus on the right side of the equilibrium ( H2CO3 <--> HCO3- + H+). The ratio of the forward and reverse rate constants (kf and kr) will give the equilibrium constant (keq), which is also called the acid dissociation constant (ka) of carbonic acid (H2CO3) in this case. In equilibrium, the forward and reverse reaction rates are equal.
• kf * [H2CO3] = k* [HCO3-] * [H+]
• ka = keq = kf / k= [HCO3-] * [H+] / [H2CO3]
The negative logarithm of kis called pKa.
• pKa = -log10([HCO3-] * [H+] / [H2CO3])
= -log10([H+]) - log10([HCO3-] / [H2CO3])
Because pH = -log10([H+]), we can rearrange the equation as below:
• pH = pKa + log10([HCO3-] / [H2CO3])
This is the famous Henderson–Hasselbalch equation. The pKa of carbonic acid (H2CO3) at 37 degrees Celsius in an aqueous solution that has physiological ionic strength is 6.1. Hence,
• pH = 6.1 + log10([HCO3-] / [H2CO3])
Now we need to rearrange this equation to relate it to the concentration of CO2. Let's focus on the left part of the equilibrium (CO2 + H2O <--> H2CO3). According to Henry's gas solubility law, concentration of COin the liquid phase is a product of partial pressure of CO2 in the gas phase and Henry's constant:
• [CO2(aq)] = pCO2 * k
Henry's constant for COat 37 degrees Celsius in an aqueous solution that has physiological ionic strength is 0.0229 molar/atm. Because our cell culture incubators maintain the partial pressure of COat a constant value, the concentration of H2COis as below:
• [H2CO3] = pCO2 (atm) * 0.0229 (molar/atm)
The partial pressure of CO2 is a product of total pressure and the percentage of COin the gas phase. The total pressure is 1 atm (unless your lab is on a mountain). Hence,
• pCO= 1 atm * %CO2 * (1/100)
• [H2CO3] = 1 atm * %CO2 * (1/100) * 0.0229 (molar/atm)
= 0.000229 * %CO2 molar
Note: %CO2 represents the number after the percentage (%) sign in this equation.
Now we write this in the Henderson–Hasselbalch equation:
• pH = 6.1 + log10([HCO3-] / (0.000229 * %CO2))
Let's rearrange this equation for our cell culture system. We have an initial amount of sodium bicarbonate (NaHCO3) in our cell culture medium. The concentration of NaHCO3 in the culture medium is also the initial concentration of HCO3- (NaHCO3 --> Na+ + HCO3-). In an equilibrium reaction, if you increase/decrease one of the reactants or products, the system will try to reverse this change until it reaches the equilibrium. So, in our buffering system, having an initial amount of HCO3- will move the reactions to the left (CO2 + H2O <--> H2CO3 <--> HCO3- + H+). An 'X' amount from the initial amount of HCO3will be used in the left direction and produce an 'X' amount of H2CO3, which in equilibrium will be equal to the amount of H2CO3 we calculated above by using the percentage of CO2 in the gas phase, which is kept constant by the incubator. So,
• pH = 6.1 + log10(([HCO3-](initial) - X) / (0.000229 * %CO2))
• X = [H2CO3] = 0.000229 * %CO2
• pH = 6.1 + log10(([HCO3-](initial) - (0.000229 * %CO2)) / (0.000229 * %CO2))
= 6.1 + log10(([HCO3-](initial) / (0.000229 * %CO2)) - 1)
Finally, [HCO3-] is a molar concentration. We want to write this in g/L. Molar mass of sodium bicarbonate (NaHCO3) is 84.007 g/mol.
• [HCO3-](initial) = [NaHCO3] = (g / (g / mol)) / L = (1/84.007) * NaHCO3(g/L) molar
So, the final equation is as below:
• pH = 6.1 + log10(((1 / (84.007 * 0.000229)) * NaHCO3(g/L) / %CO2) - 1)
= 6.1 + log10((51.98 * NaHCO3(g/L) / %CO2) - 1)
PS: In my previous post, I did not take into account the amount of sodium bicarbonate present in FBS when calculating the final concentration of sodium bicarbonate in the culture medium upon addition of FBS and L-glutamine. FBS contains around 2 g/L of sodium bicarbonate. Check the lot specific certificate of analysis of your serum to find the exact value, if possible.
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Is sodium bicarbonate installation (small amount and diluted) used for artificial airway (ETT and Tracheostomy) suctioning to remove thick secretions? Clinically and theoretically, Why and why not?
Intensivists, pulmonagists, Critical Care Nurses and researchers.
Thank you.
As far back as 2008 evidence identified the instillation of normal saline before endotracheal suctioning has been considered an adverse practice because of the physiological and psychological effects. More current evidence has reached similar conclusions.
This review article by Halm et al may be initially helpful: Halm et al, Instillingn normal saline with suctioning. Am J Crit Care September 2008 vol. 17 no. 5 469-472
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A 3.5% saline solution prepared with water and sodium has a pH of 6.38.
I want to make a solution of 3.5% salinity with a pH of 8.1 how do i do this using only sodium, and sodium bicarbonate?
I need to adjust for the sodium of the sodium bicarbonate in regards to the salinity whilst maintining a ph of 8.1 and salinity of 3.5%.
The aim of this is to make a solution that will resemble sea water pH and salinity.
Thanks Guido Bongi sir!
It's the most pertinent information.
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Hi! This protocol has been used in my lab to handle healthy rat liver samples (see below). But now, i'm going to start a project for quantifying membrane proteins in healthy and diseased human liver tissue and this doesn't feel like a safe way of doing it.
Has someone homogenized Human liver before that could share a few tips and maybe the protocol they followed?
thank you!!!
Isolation of membrane protein
Rat liver and kidney weighing were homogenized using glass dounce homogenizers in Sodium Bicarbonate with protease inhibitors. After brief centrifugation, the clear supernatant was incubated with Sodium Carbonate at 4 oC for 15 minutes. Samples were centrifuged at 100,000g for 1 hour at 4 oC. The pellet was resuspended in1xPBS containing protease inhibitors and disrupted briefly by ultrasonication. The protein was quantitated by Pierce BCA Protein Assay kit.
Steingrimur,
Thank you so much for the info sheet!  I already contacted bio-safety office but definitely this helps.
Also I found a way to homogenize liver in a safer way than using the dounce homogenizer. If I get good results I'll posted them here just in case someone's interested.
Best, A
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Addition of sodium bicarbonate to the medium alters the pH of the broth and by which the growth and production of microbe gets altered; but , does it really alter the structural composition of the PHA synthesized?
Some authors studied the effect of pH in PHA production,and they observed that PHA composition was strongly affected by the pH; HV fraction increased with the pH increase in the media.
If you are interested in knowing more about this I recommend you to read the article: "Effect of pH on the production of bacteria polyhydroxyalkanoates by mixed cultures enriched under periodic feeding" written by Villano et al. 2010.
Best regards.
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For herbal antacid/effervescent , will sajjikshar and lemon juice powder will give the same amount of fizz as given by lab grade citric acid and sodium bicarbonate?
@Asmita Wele
OK mam
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1. I intend to prepare a 10 mM solution of reduced glutahione by dissolving in DMSO.
2. Further, I plan to take an aliquot of the above stock and dissolve in alkaline water (prepared by adding 8.4 g sodium bicarbonate / liter of distilled water).
3. I do not want it to oxidize at least for a couple of hrs. after diluting.
I know GSH is stable in PBS pH 6.5 with 1 mM EDTA, and is more stable in alcaline conditions. Sorry I can´t help more :)
You can also use a nitrogen flow to avoid oxidation, which is more guaranteed.
Cheers!
Rachel
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My cell line is recommended to be grown in 4mM glutamine,4500mg/l glucose and 1500mg/l sodium bicarbonate but I am growing in 2mM glutamine,2000mg/L glucose and 2000mg/l bicarbonate. will it affect the growth of my cell??
For instance, MCF-7 is recommended to grow in EMEM and 0.01mg/ml of Insulin by ATCC. But, I observed in many papers that MCF-7 growing in MEM/RPMI/DMEM(glucose levels 1g, 2g, 4.5g per liter, phenol red quantities varies respectively etc.). To avoid confusion its better to stick to cell line source composition.
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1.Can we use Oxidise -Redox (GSH:GSSG) system for sodium bicarbonate as a folding buffer?
2. If we use, what will be the impact during citraconilation and Trypsinisation?
Yes, that redox couple can and has been used but be open to using other redox couples and testing a range of ratios of ox:red forms.  Be sure to allow your refold to go to completion where it is negative to the Ellman's test to ensure no interference with running your anhydride reaction and running non-reduced SDS-PAGE.  Going to completion may take relatively long or short times dependent upon the protein and refold system.  Predicting the completion time is dependent upon the kLa (coefficient of mass transfer between gas/liquid interface) involving temperature, oxygen solubility in your buffer system, Reynolds number, fluid depth, vessel morphology, impeller design, wattage, etc.  The pKa of the glutathione thiol should also be compatible with your bicarbonate buffer which implies that you will be working between ~pH 9-11, maybe pH 9.  Your maleimide and amide formation with K's, R's and N-term should be fine at this pH with all the glutathione oxidized which won't affect the trypsin.   Lack of cleavage should then be representative of the blocking.  If you want to reverse it at acid pH to prove the point, the glutathione will not be affected but watch for CO2 bubbles.
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For preparing DMEM media, I’ve used DMEM powder with neither HEPES nor sodium bicarbonate. As a buffering system, I added HEPES to the solution to the final concentration of 20 mM and adjusted the pH of the prepared media to 7.3
Hi Sara,
It depends on what are you using the medium for. If you are using the medium for cell culture in a CO2 incubator, sodium bicarbonate is a buffering reagent provided 3-5% CO2. Supplemented with HEPES, gives you a better buffering capacity of the medium, if your cells require a higher range of pH (7.3-7.6).  So it really depends on your cells as well. I would recommend a side by side comparison of two medium with or without bicarbonate to see the difference.
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Hi everyone!
I am trying to culture cells from ATCC (ref: CRL-1520-ATCC) in order to obtain the A2B5 antibody. As the company recommends, I use a DMEM formulation from Sigma-Aldrich (D-7777) with a sodium bicarbonate concentration of 1.5g/L.
I did not use antibiotics as it was not specifically recommended and it was the first time of culture so no contamination was expected. However, after two weeks of culture the cells were full of bacteria.
Does anybody know the best way to culture this cell line? Has anybody used any different media from the ATCC DMEM?
Hi
You must use antibiotic in recommended dose. i am agree with above said suggestions. Few more suggestions are antibiotics should not be used routinely in cell culture, because their continuous use encourages the development of antibiotic resistant strains and allows low-level contamination to persist, which can develop into full-scale contamination once the antibiotic is removed from media. Further, some antibiotics might cross react with the cells and interfere with the cellular processes under investigation.
Antibiotics should only be used as a last resort and only for short term applications, and they should be removed from the culture as soon as possible.  If they are used in the long term, antibiotic-free cultures should be maintained in parallel as a control for cryptic infections.
Hope this helps little
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I understand that it depends on the cell type. Mine is MacT cells, pretty stable mammary cell line. The papers that I read the longest will be 12h after switching from basal medium (serum+, glucose+ DMEM)
I know it will be better to set a time-response experiment first but I need to set a reasonable time scale and save time...My goal is just to compare the effect with or without glucose...
Plus, The glucose free DMEM that I have is D5030 sigma, no glucose, no phenol red, no L-glutamine.
My intended preparation protocol was,
Dissolve the glucose free DMEM, filter, add antibiotics,
I wonder if I need to add sodium bicarbonate as what other DMEM medium does...and serum and L-glutamine?
My best guess was, no serum, no glucose, maybe plus plasmid transfection. The cells will not survive very long....
If you use plasmid transfection, your cell should be in a good and proliferative stage. So i strongly suggest that wait after transfection for a duplication time at least to get proper plasmid response in cells. L-glu is essential, and bicarbonate you need for pH. If you would like to starve your cell, do not add glucose, and a minimal FCS only. Strongest starvation medium even is lack of AAs. and cell lines rather tolerate in that only 1-2 hours.
You need a normal basal medium, and this one you mention. Transfect in basal, than change the medium (you can split (passage) the transfected cells from a bigger pool, but plate them in basal medium for a while) to the mentioned one, with combined glucose/FCS in different manner first.
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can i use sodium bicarbonate? or there any better preservatives?
Hi Irwan,
Following updated review might be helpful for you.
Regards,
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I am working on capillary monolith cation exchanger suppressor using 1.75mM sodium bicarbonate/ 1.5mM sodium carbonate with Contactless Conductivity detector. The analyte concentration is 1.5mM
1. Anion sample is dissolved in pure water.
I get the point to use simpler eluent and try to apply it.
The literature does help too.
2. No, the retention time of the peaks was slightly different. For example, 1st injection was 7.03 minute for NaF, second injection was a bit longer 7.07 minute.
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I am using hydrazine but I have to extract unreacted hydrazine into sodium bicarbonate. There I lose my alkoxyamine a lot.