DMSO - Science topic

Hello Shaokai Pei

Use 0.2M sucrose phosphate as a cryoprotectant for varicella-zoster virus.

You may want to refer to the article attached below.

However, virus infected cells may be frozen in 70% culture media + 20% FBS + 10% DMSO.

Best.

There is no fixed relation between solvent and edge specific of GQDs , the edge frame synthesis regards to methodology and precursor you intimate with !

My idea here would be to thaw the FBS to make aliquots by mixing it with the DMSO (and maybe PMEM) and then again to use it to freeze the cells - it should be fine, right?

the highest DMSO concentration (0.1%) should be sufficient as a control since it'll include potential sides effects of lower concentrated vehicle

I'd suggest to try 90%FCS and 10% DMSO (v/v)

Obtain phloretin (Sigma-Aldrich P7912-25MG). Resuspend 25mg in 1.825ml DMSO to bring to (1000x) 50uM stock concentration. Aliquot 100ul and store at -20 C (good for 1 month). Aside: also dissolvable in 30%DMSO in PBS (however this will only be good for ~1 day) Aside Aside: you can also disolve in EtOH at 10mg/ml

That's good to know! Thank you!

try transcutol

or

John Patrick Dodson I was curious if you had any updates.

Martin T. Bosnev Thank you so much!

Hi Aroona Razzaq,

You may use the web tool to calculate

Dear Saket Phadkule, my answer is a general comment since no details are supplied about the environment to which DMSO is in contact. DMSO is a very reactive solvent, which means possible chemical changes occur upon contact with other sources such as compounds with reactive hydrogen, metals, halogens .... The most unpleasant problem is that it is a solvent that may generate peroxides under known conditions. Peroxides in spite of their toxicity, they are extremely reactive, may provoke a runaway and violently reaction. So, if it is to be reused, one may expect surprises. Page 8 in the attached document shed light on some features. My Regards

There is a whole chapter on effects of solvents on florescence emission spectra in Joseph Lakowicz's textbook "Principles of Fluorescence Spectroscopy" (Chapter 7 in the 1st edition). He divides solvent effects into general and specific types. The following is an excerpt from p. 189.

"By general solvent effects we mean those which result from the refractive index (n) and dielectric constant (epsilon)....Specific solvent effects refer to specific chemical interactions between the fluorophore and the solvent molecule, such as hydrogen bonding and complexation."

The rest of the chapter goes into details.

Muhammad Nouman Shaukat hello, your recommendation is good to receive a concentrated extract. but in my country, a test to show no ethanol is available in the food is necessary. moreover, ethanol in even low concentration would affect microbial growth and probably sensory parameters of dairy products.

It dissolves the cell membrane.

As pointed out by Jeremy Pronchik , it is not necessarily true that a given protein molecule is either fully active and in its native conformation or fully inactive and unfolded. A protein molecule's structure might be mildly perturbed by a low concentration of DMSO and retain partial activity. High solvent concentrations, on the other hand, would probably result in major loss of native structure of most proteins.

If a solution of a protein at a reasonably high protein concentration is substantially unfolded, it will almost certainly result in aggregation of protein molecules due to exposure of the hydrophobic interior. In this extreme case, a simple measurement by dynamic light scattering will reveal the presence of aggregated protein. This requires a special instrument, but if the aggregation is severe enough, it can even be detected with a UV-VIS spectrophotometer, which most labs have.

The effect of a solvent or other chaotrope on the stability of a protein can be monitored by measuring the melting temperature of the protein in solution, using either differential scanning calorimetry, or differential scanning fluorescence. Each measurement requires a special instrument.

Many proteins contain tryptophan residues whose fluorescence is sensitive to their environment, specifically the hydrophobicity of their environment. Using a spectrofluorimeter, you can measure the tryptophan emission spectrum to follow major and minor structural changes in the protein. This can also be done using far UV circular dichroism (CD).

Far UV CD, mentioned by Jeremy Pronchik is used to measure changes in the secondary structure elements of the protein. Loss of secondary structure would be a sign of a major loss of native structure. I think the presence of DMSO would interfere with this measurement, however.

@Malcolm Nobre thank you for your response. Yes, I preserved the cells via Mr.frosty at -80, and also carried out the quick thaw process. But I didn't maintain a temperature log for the -80 freezer

DMSO most often helps to clean up multiple amplimers so that only the correct one amplifies but if a hotstart taq does not help than I do not think that dmso will help but it is worth a try

It is a worry that using less primer causes the control to fail as it suggests that either your primers are very prone to dimerisation or that you have some contamination of primer dimer in one of your reagents. PD contains both primer sequences and is very short so amplifies very well ( melts easily, efficiently binds primer and being short it amplifies very well).

Primers are always present in large excess but it sounds like you are removing too much primer as primer dimer so if possible redesign your primers using a program like primer3/3+ which minimises the possibility of PD. Primers are cheap and your time and peace of mind suggest to me that new primers will help and will also give you the possibility of using old and new primers mix giving an increased chance of a clean amplification

To achieve a final concentration of 0.1% DMSO while maintaining the desired concentration of the plant extract, you can follow these steps:

Here's a summary of the steps:

i think you can also use a high anneling with betain solution.

You're right, adding ammonium sulfate could potentially quaternize the tertiary amine groups in your polymer, which could affect the properties of your anionic exchange membrane. If you're concerned about altering the chemical nature of the polymer, it's best to avoid using salts that could react with it.

In that case, you might want to focus on solvent-based methods for precipitation. You could try experimenting with different solvent mixtures or adjusting the temperature of your solutions to see if that helps with the precipitation without chemically altering your polymer.

Dear Ismail Khan, please have a look at the following RG link, may be it gives a partial answer. What is the nonachieved expected result by your recipe. My Regards

https://www.researchgate.net/publication/230494097_Role_of_dimethyl_sulfoxide_as_a_solvent_for_vinyl_polymerization#:~:text=They claimed that DMSO reduces,the polymer structure took place.

try amplifying in final concentration of betaine 1molar and 5%dmso ( up to 8% is ok with pcr) using primers far apart then if this looks poorly amplified dilute the amplified product 1:100 and re amplify with any primer pair inside the first pair of primers also using betaine and dmso. Check the quality and quantity of your original dna sample in case it contains pcr inhibitors and try amplifying double dilutions ( 1/2, 1/4, 1/8 etc) as sometimes diluting a pcr inhibitor means better amplification even though there is less dna

Hello, could you please tell me how you solve this problem?

PSCs shouldn't be stored in -80C for more than a week. If storing for 2-4+ years, definitely transfer them to liquid nitrogen. It also helps to place your cryovials in a "Mr. Frosty" (or equivalent box), filled with isopropanol, to help the cells freeze at a controlled rate of -1C/min at -80C overnight before transferring to liquid nitrogen. I've noticed that it does in fact increase post-thaw viability alongside ROCKi. I've thawed hiPSCs, frozen in liquid nitrogen for 3+ years, and have had no issue.

Also, storing PSCs in serum will cause them to lose their pluripotency. And much like your case, a colleague of mine stored his with FBS (in his case he was using hESCs and hESC-derived NPCs, and they did in fact clump as well) and they failed to proliferate before completely detaching from the plate after a few days.

As I understand it, serum push PSCs to differentiate, and doesn't sit well with their respective media which helps them maintain their pluripotency and self-renewal.

I'd recommend using Knock-Out Serum Replacement, as opposed to serum, or using PSC Cryomedium (links to both below). They both work really well with my hiPSCs and hNPCs, as well as for my colleagues' human and mouse ESCs.

Hope this helps.

2,4-Dinitrophenol (DNP) is sparingly soluble in water and moderately soluble in DMSO (Dimethyl sulfoxide). If you want to dissolve it all in the vial it came in, DMSO might be a better choice for higher solubility compared to water. However, always follow safety guidelines and ensure compatibility with your intended use.

Hello, I am also trying to find the answer to this. Did you ever end-up dissolving it?

Hello, Steven Choi

Although I haven't synthesized Perovskites with Cs or Cl, I have been working with CH3 cation and Iodine anion. DMSO can be used to dissolve my precursors, but also Dimethylformamide (DMF), and gamma-butyrolactone.

Mistures of DMF and DMSO also have been reported to dissolve them too.

Best regards,

Ricardo Tadeu.

Dinitrophenol is quite stable so you probably could even store at room temperature if not exposed to the light. A general rule of thumb is 1 month for 4°C, 6 months for -20°C and 2 years for -80°C. For your other two compounds I would suggest storing as single use aliquots at -20°C.

If you really want to do a classic antibiotic testing then I concur with Adam B Shapiro although I would recommend doing the assay as a MIC 50 assay in liquid culture. I worry that with plates if the substance is not at all diffusing then the interpretation of the results may be problematic.

However I am not sure if this is really the right approach at all for a material that is likely to be used on a surface. At least I assume that is the intention otherwise why develop and insoluble agent. There should be a literature on developing antimicrobial surfaces and those would likely have procedures.

If the ligand-into-buffer thermogram is significantly different to the ligand-into-protein thermogram, then, as a first approximation, you may subtract the first from the second thermogram. This will provide you the net heat effect "mostly" coming from the interaction. However, this is an approximation since the ligand self-dissociation will be coupled to the interaction with the protein and will result in an apparent interaction affinity and enthalpy that do not correspond to the intrinsic interaction parameters. However, if you are just concerned about demonstrating there is interaction, that is fine, and, at least, you have some approximate estimation for the interaction parameters.

A more appropriate alternative would be determining the self-association parameters from the ligand-into-buffer thermogram, and then develop a model in which ligand dissociation is coupled to the protein binding. Using the self-association parameters as known parameters, you may determine the protein binding parameters.

Hello Alessandra,

I also use a 10% DMSO/90% FCS freezing solution, to some extent the quality of the thaw correlates with that of the freezing process. I find that a cooling rate of ~-1˚C/min in a (Mr Frosty) or CoolCell in a -80˚C freezer, before transfer to liquid nitrogen for long term storage is best for sensitive cells.

1. allow appropriate culture medium to warm to room temperature.

2. thaw cells in 37˚C waterbath, with gentle mixing, until a only a single small ice crystal remaining.

3. immediately transfer cells to 50ml polypropylene conical tube.

4. add 1ml complete culture medium dropwise - mix by swirling, leave for about 30 seconds, to allow DMSO to diffuse.

5. add 2ml complete culture medium dropwise - mix by swirling, leave for about 30 seconds.

6. repeat adding doubling volumes until 31ml complete culture medium added.

7. centrifuge 7 minutes at 400g.

8. resuspend pellet in 30ml complete culture medium.

9. repeat centrifugation.

10. resuspend cells at desired density for use.

Malcolm has already pointed out not the importance of NOT leaving the cryovials unattended during the Thawing process.

Best wishes

May be helpful

Addition of scavenger or antioxidants to minimize oxidation. For instance, small amounts of molecular sieves or a radical scavenger like TEMPO (2,2,6,6-Tetramethylpiperidine 1-oxyl) can be added.

Control the reaction temperature to moderate levels. Elevated temperatures can contribute to undesired side reactions, including oxidation.

Adeola Folasade Odewusi-Ehigie

In this case, particulate contamination was not an issue at all. Since oil is immiscible with many liquids and usually forms a separate layer on top, we were searching for a solvent to form a homogeneous mixture.

DMSO as a solvent and sonication were helpful.

Thank you for sharing this valuable information.

Regards.

Please have a look at this previous thread

Some people are suggesting ethanol or methanol.

Thank you Dear Dr Balamurugan

Dear Sheila D Shay

You may have got the right information. You may want to refer to the article attached below for more information.

Regards,

Malcolm Nobre

Follow the following steps

Select a DPP-4 Inhibitor: Commonly used DPP-4 inhibitors include Sitagliptin, Vildagliptin, or Linagliptin. Choose one based on availability and compatibility with your analysis method.Prepare Stock Solution: Dissolve the chosen DPP-4 inhibitor in an appropriate solvent to create a stock solution. Ensure the concentration is suitable for your experiment.Add DPP-4 Inhibitor to Blood Samples: Mix the stock solution with the blood samples immediately after collection. The concentration of the DPP-4 inhibitor should be sufficient to inhibit the enzymatic activity effectively.Mix Gently: Ensure thorough mixing of the DPP-4 inhibitor with the blood samples to distribute it uniformly.Incubation: Incubate the samples for a specified time at an appropriate temperature to allow the inhibitor to act on DPP-4 and inhibit GLP-1 degradation.Centrifugation: Centrifuge the samples to separate plasma or serum from cellular components.Store Properly: Store the treated samples at the recommended temperature and conditions to maintain the stability of GLP-1 until analysis.

Hello Rasha Sl

PEG 400 is well tolerated in mice at a dose of 10mL/kg IP at 35% for 3 days or at a dose of 2.5mL/kg IP at 40% for 1 month. You may want to refer to the article attached below for more information. Please see Table 75 (PEG 400) under mouse section.

Best.

@Malcom Nobre.

Dear Dr Nobre I checked the process of cell freezing and figured out that the -80 cold room had been out of order for a day. I think the problem was related to cold room.

I like Miao Zheng's idea, but with one change. I recommend you pre-test with only a negative control (zero sap). You want to observe any effects on your cell line produced by the highest concentration of DMSO you will test. To get that number divide your highest sap concentration sample by your stock concentration: 100 ug/mL / 10 mg/mL = 0.01, or 1%. So I recommend you start by testing 100 ul of a 1% solution of DMSO (diluted in your growth or harvest medium) against your cells. DMSO generally is not well-tolerated by cell cultures as it dissolves cell membranes, but you may be able to use it in a brief assay. You need to know about any changes to your cells. So check the DMSO-exposed cells in your assay, but also look at them under the microscope. If they seem "normal," we can set up a simple dilution series for your test samples.

Let me warn you that making dilutions of DMSO into water is tricky, because DMSO dissolves in water (about 70%). If you do end up using DMSO as your carrier solvent I recommend you prove this awkward fact to yourself by mixing 10 mL of DMSO to 10 mL of water and then measuring the final volume in a graduated cylinder. It is not 20 mL, as you may expect; it's closer to 13 mL. So if you use DMSO for your stock solution you'll need to account for this phenomenon when you make your dilutions. For samples at low concentrations the effect will be insignificant, but for higher concentrations you will want to estimate the effect to avoid a large systematic error.

Hey there Neha Bisht! So, you're diving into some serious chemistry, huh? Well, here's the deal with GeI2 powder – it's a bit of a stubborn one. Seems like it's giving you Neha Bisht a hard time dissolving in the usual suspects.

Now, considering you've tried DMF, DMSO, NMP, and even ethanol, and they all resulted in a cloudy solution, I'd say we need to step up our game. How about giving a shot to something more exotic? Have you Neha Bisht considered trying out THF (tetrahydrofuran) or acetone? Sometimes, these outliers can surprise you Neha Bisht.

But hey, chemistry is all about experimentation, right? So, roll up your sleeves, break some rules, and let's see if we can coax that GeI2 into playing nice. Good luck, and don't let those cloudy solutions rain on your parade!

It seems that your template concentration is way too high and the algorithm doing the baseline correction is confused/not working properly.

I guess it would help to dilute your samples by a factor of 1 to 10 million.

Hello Prashsti Chadha

There are two schools of thought when it comes to removing the cryoprotectant from the cells during the process of thawing.

1. Generally, the cell suspension is centrifuged and the supernatant containing the cryoprotectant (DMSO) is discarded, and the cell pellet is resuspended in fresh cell growth medium prior to placing the cells in the culture vessel.

2. However, in case of fragile cells, you need not centrifuge after thawing because the centrifugation process may be detrimental to the cells thereby increasing cell death. Therefore, in such a case, it is recommended that the cells be plated without centrifugation and that the growth medium be changed after 24 hours or even earlier once the cells have attached to the substratum to get rid of the cryoprotectant.

I would suggest that you thaw SH-SY5Y cells just before you are done for the day, leave the cells in the incubator for the night and change the medium with fresh medium first thing the next morning.

The freezing medium for SH-SY5Y cells is complete growth medium containing 10% FBS and supplemented with 5% DMSO. You may also use freezing medium containing 95% FBS and 5% DMSO.

Whenever you freeze mammalian cells, you should follow the gradual freezing technique (at the rate of -1°C /min) by using Mr. Frosty freezing container which is a system designed to achieve a rate of cooling very close to -1°C/min, the optimal rate for cell preservation. During the freezing process, after transferring 1 ml resuspend cells in each vial, keep vials at 4°C for 10 minutes, then at -20°C for further 10 minutes. Then move the cells to -80°C overnight and then transfer the vials for final storage in liquid nitrogen the next day.

Also, you should harvest the cells during their maximum growth phase (or log phase) and should have greater than 80% confluency before freezing. Additionally, it is important to consider the optimal concentration for freezing cells which may vary depending on cell type. Typically, the concentration of cells in the cryogenic vial should be within a general range of 1x10^5 - 1x10^6 cells/ml. If these above factors are taken into consideration, you may be able to store the cells in liquid nitrogen for several years.

Best.

EVA it is not soluble in polar solvents, that is why it is sold in Toluene. The only reference that I have it is that is soluble as much as 8% concentration of anhydrous dimethyl sulfoxide (DMSO).

Best regards

Frank Schwoebel thank you so much

Add crown ether, dicyclohexyl-18-crown-6

The stuff is insoluble in water. The best you can do is a suspension.

Option A: You can try a ketones (acetone, MEK, etc)

Option B: You can make a prepolymer at 100% solid and dilute later for further reactions

Hi,

You may use the web tool to calculate

Adam K A Wright Bambanker is designed to specifically avoid the use of a controlled-rate freezing apparatus (such as an isopropanol container). The use of such an apparatus will negatively affect the viability. Instead, follow the protocol. Just add the media to cells and immediately place into -80C.

Dear friend Schahrazede Lacheheb

Ah, the noble pursuit of refining algae extracts for NMR characterization! Now, let me guide you Schahrazede Lacheheb through this endeavor with flair.

Firstly, dimethyl sulfoxide (DMSO) can indeed be a pesky companion, but fear not, I have some wisdom to share:

1. **Simple Evaporation:**

- One straightforward method is to evaporate DMSO by exposing your algae extract to air. This can be done using a rotary evaporator or simply by leaving it in an open container. However, this might take some time.

2. **Vacuum Filtration:**

- Use a vacuum filtration setup to remove DMSO. Apply a vacuum to speed up the process. This can be effective for separating the solvent from your algae extract.

3. **Liquid-Liquid Extraction:**

- Consider liquid-liquid extraction with a less polar solvent, like ethyl acetate or diethyl ether. This can help to partition the DMSO into the less polar phase, leaving your algae components in the aqueous phase.

4. **Chromatography Techniques:**

- Chromatography methods, such as column chromatography or solid-phase extraction, can be employed to separate DMSO from your algae extract.

5. **Diafiltration:**

- Use diafiltration techniques, like ultrafiltration or dialysis, to selectively remove DMSO based on differences in molecular weight and size.

Remember, my advice is bold and daring, but practical considerations should guide your choice. The method you Schahrazede Lacheheb select depends on the characteristics of your algae extract and the equipment at your disposal.

Feel power coursing through your veins as you Schahrazede Lacheheb embark on this extraction quest! May your NMR characterization be as pristine as an unbounded wisdom!

Spectrophotometers have limits to how high an absorbance they can measure accurately. Depending on the instrument, this may be as low as 1 or as high as 3. If you are struggling to measure the absorbance of your peptide because of high background absorbance, one solution is to use a shorter pathlength. Remember that absorbance is directly proportional to the pathlength of light through the sample. If you are using a standard 1-cm-pathlength cuvette and get an absorbance of 2, if you switch to a 0.2-cm-pathlength cuvette, the absorbance will be 0.4, well within the useful range.

I think DMF may work better.

Fotonlar çift çekirdekli kabuk olan altın - gümüş için ayrı ayrı gönderilebilir(ayrı ayrı kaplama ve katlama yapılarak) veya bir alaşım (altın - gümüş) oluşturularak kaplanarak foton gönderilebilir. Bu sırada laser ablasyondan çıkan fotonlar için çift atış yapılmalı, çekirdek ve lazer kaynağı arasında foton için iletim, taşınım ve yayılım sağlanmalıdır.

Dear Dr. Maral Tabrizi

The protocol that you have mentioned to fix cryopreserved cells using 10%NBF may be damaging to cells. The cells will undergo a lot of stress as you will be subjecting them to many treatments like thawing, washing, centrifugation and fixation within a short period without giving the cells enough time to recover. This is going to be stressful to the cells.

I would recommend that you first thaw the cells by a quick thaw method, culture the cells for 2-3 passages, trypsinize the cells on confluency, wash once with PBS, and resuspended in approximately 150-200 μl of 10% neutral buffered formalin (NBF).

You may transfer the resuspended cells in a microcentrifuge tube with solidified 2% agarose prefilled in the tapered portion of the bottom, and spin down at 540 ×g for 5 minutes. The resulting supernatant may be removed, and you may add fresh 10% NBF without disturbing the pellet. The microcentrifuge tube may be spun at 810 ×g for 5 minutes, and the resulting cell plug may be fixed in formalin for 48 hours at room temperature by submerging the microcentrifuge tube into a 15ml conical tube containing 10% NBF.

After fixation, the microcentrifuge tube may be transferred to a new 15ml conical tube with PBS and stored at 4°C prior to processing and embedding in paraffin blocks.

I feel this method would be more appropriate than fixing the cryopreserved cells.

Best.

Dear Naily,

You have to consider first the solubility of TQ in DMSO, after that you can go to diluting DMSO.

Depends on the route of administration. For I.P I have used up to 10% with no adverse toxicological effects. For parenteral administration I would not go above 5%. All this is for experimental animals, no idea in humans...

0.050 gr IBUPROFEN in 1 gram of DMSO seems to be fairly concentrated to me, but it depends of the temperature. At room temp it may be OK but keep it above 5°C or DMSO will crystallyze.

Pubchem is good place to start

Further publications

Can you dissolve PLA in NMP to get a saturated solution?

Dear Agnė Keselytė,

First of all, I would like to inform you that the best method for DMSO analysis is Gas Chromatograph (GC) because having solvent this technique is best suitable.

Gene@rally, DMSO is not analysis by HPLC because the traces can be remain in system and DMSO will cause corrosion.

Thank you so much Malcolm Nobre

If you perform your SEC purification with organic solvents as eluent, I prefer to use Sephadex LH-20. This resin can withstand efficiently with organic solvents from my own experience.

IMQ is a molecule highly insoluble in aqueous media; its solubility increases slightly at acidic pH because it is a weak base (pKa = 7.3). Solubility of IMQ in DMSO is 1.29 ± 0.13 mg/ml (at room temperature, under magnetic stirring).

Hello Dorian Foster

if you can add some data files it might help here.

Did the manufacturer state any suitable conditions for the material, then that might be a good starting point.

Colloidal Gold particles can be quite monodisperse, and are even available as a standard, see https://www.materials-talks.com/polydispersity-from-a-gold-standard/ and this poster

For more generic nanoparticle characterization topics you might like this "question & answer" collection at https://www.materials-talks.com/tips-tricks-for-nanoparticle-characterization/

Yes, you just need proper controls. Enzymes will have different tolerances for DMSO, and so the first step will be to measure your enzyme rate at different DMSO concentrations. Once you know the maximum DMSO tolerated without loss of activity/denaturing, you can design your inhibition assay. If your enzyme loses a lot of activity or denatures at these DMSO concentrations, you might have to try other solvents until you find something compatible.

For example, I worked with an enzyme that retained 100% activity up to 7% v/v DMSO and then started precipitating near 10% v/v. I then wanted to test an inhibitor from 1 mM to 1 µM so I prepared 20X stocks of each inhibitor concentration (20 mM to 20 µM) in 100% DMSO. From these stocks I added the inhibitor to the enzyme to a final concentration of 5% v/v, or 1:20 dilution and the inhibitor did not precipitate. The positive control receives just 5% v/v DMSO with no inhibitor. Using this design you can confidently measure the effect of the inhibitor while controlling for the effect of DMSO consistently across replicates.

Hope this helps!

ACA

Thanks, Jonnalagadda Bhavana.

I am still facing that problem. Can anyone please help?

DMSO can significantly decrease fluorescence intensity in Fluo-4 based calcium assays. This is likely due to:

- DMSO is hygroscopic and absorbs water from the assay buffer, which can reduce Fluo-4 fluorescence intensity.

- DMSO can directly interact with Fluo-4 and cause fluorescence quenching through various mechanisms.

- High concentrations of DMSO can disrupt cell membranes and calcium channels, reducing the influx of calcium upon ATP stimulation.

- DMSO is amphiphilic and can bind to hydrophobic pockets on proteins, altering their confirmation and function.

To avoid these interferences, an alternative solvent such as ethanol or methanol can be used to dissolve compounds for calcium assays.

And appropriate vehicle controls should also be included.

Thank you for answering! Dhiraj Kumar

Dear Mansur Dabai Salisu

You may dissolve 51.2mg Palmitic acid (mol.wt. of Palmitic acid is 256.43 g/mol) in 1ml ethanol (100%) to give a stock of 200mM.

Take 40ul of the above stock solution and add to 1.96ml of 10% fatty acid free BSA (in culture media) to give 4mM diluted stock of Palmitic acid. The solution must be shaken overnight at room temperature.

The following day, you may filter sterilize the diluted stock using 0.22μM filter. If required, further dilutions to be used in cell treatment may be made in culture media.

Best.

It is free dissolve in ethanol.

Hello Robson Araujo

Sorry! I did not understand the calculations from the excel sheet as it is very complicated.

“Could someone help me to do the dilution directly?”

Yes, let me make it simple.

The Molecular weight of the compound (C23N3OH27) is 361.48.

Then follow the sequence below.

361.48g -------- 1L -------- 1M

361.48g --------- 1L ------- 1000mM

0.36148g ---------- 1L ------ 1mM

361.48mg -------- 1000ml ------ 1mM

3.614 mg ----------- 10ml -------- 1mM

So, weigh 3.614mg of the compound in 10ml 100% DMSO to give 1mM stock.

You may prepare working solutions (30uM, 20uM, 10uM, 5uM, 1uM) as follows.

You may use the formula: C1V1=C2V2

C1= Concentration of stock solution (1mM)

V1= Volume of stock solution (X)

C2= Concentration of working solution (30uM)

V2= Volume of working solution (say 1ml)

Then,

1mM x X = 30uM x 1ml

1000uM x X = 30uM x 1ml

30/1000 = 0.03ml of stock i.e., add 30ul of stock solution to 970ul of media to give 1ml of 30uM working solution.

Similarly,

For 20uM

20/1000 = 0.02 ml of stock i.e., add 20ul of stock solution to 980ul of media to give 1ml of 20uM working solution.

For 10uM

10/1000= 0.01ml of stock i.e., add 10ul of stock solution to 990ul of media to give 1ml of 10uM working solution.

For 5uM

5/1000 = 0.005ml of stock i.e., add 5ul of stock solution to 995ul of media to give 1ml of 5uM working solution.

For 1uM

1/1000= 0.001ml of stock i.e., add 1ul of stock solution to 999ul of media to give 1ml of 1uM working solution.

Since 1ul is a very minute quantity to pipette, it may lead to error. So, you may dilute the stock by 1:10 to make a diluted stock (0.1mM). Then take 10ul of diluted stock (0.1mM) and add to 990ul of media to obtain 1uM working solution. Use this calculation for 1uM working solution instead of the above.

Best.

Even Xu Thank u. But, I'm working with chemical polymers.

No, the concentration of the plant extract will not be the same if the volume decreases from 500 microliters to 100 microliters while keeping the initial concentration constant at 30 mg/ml.

To calculate the new concentration after the volume change, you can use the formula for concentration:

C1 * V1 = C2 * V2

Where: C1 = initial concentration (30 mg/ml) V1 = initial volume (500 microliters) C2 = final concentration (unknown) V2 = final volume (100 microliters)

30 mg/ml * 500 microliters = C2 * 100 microliters

C2 = (30 mg/ml * 500 microliters) / 100 microliters

C2 = 15000 mg/ml / 100 microliters

C2 = 150 mg/ml

So, when the volume decreases from 500 microliters to 100 microliters while maintaining the initial concentration of 30 mg/ml, the new concentration will be 150 mg/ml.

NOTE= "If there were no physical factors involved, or you can prepare a fresh solution."

Sept. 5, 2023

Dear Theopisti,

Sorry for the delay in getting back to you.

Without more detail as to what you are experiencing and knowing exact concentrations of the reagents you are using, I guess I am at a loss to suggest a solution. However, others have had issues with this assay protocol and sought help on Research Gate. I have copied recipes and methodologies that have been submitted on this subject. They are shown below (4 responses).

I hope this information helps you.

Bill Colonna Iowa State University, Ames, IA [email protected]

Hello! I haven't personally used this recipe (http://www.protocol-online.org/biology-forums-2/posts/6375.html) but you might want to give it a try:

Mix 24 ml of 0.2 M dibasic sodium phosphate (dissolve 1.78 g Na2HPO4-2H2O or 3.58 g of Na2HPO4•12H2O in dH2O and fill up to 50 ml) with 26 ml 0.1 M citric acid (2.10 g citric acid monohydrate in dH2O and make to 100 ml with dH2O), check pH and dissolve 0.02 g sodium perborate. Store in polyethylene bottle at 4 °C stable 1 year.

Dissolve 240 mg 3,3’,5,5’–tetramethylbenzidine (m.w. 240) in 10 ml of DMSO. Store in opaque polyethylene bottle at 4 °C stable 1 year.

Mix 25 µl of TMB substrate to 1.0 ml of phosphate-citrate buffer plus perborate, stable for several hours at room temperature or for few days at 4 °C.

Protocol Available in: http://www.ihcworld.com/_protocols/chromogen_substrates/POD_TMB_blue.htm

Protocol for TMB Peroxidase Substrate Solution TMB Peroxidase Substrate  Solution –  Blue

Final Dilution: 0.005% TMB - 0.006% H2O2 in 0.01M Acetate Buffer and 0.05% Sodium Nitroprusside – toxic (or Sodium Nitroferricyanide – less toxic), pH3.3

Stock Solutions:  0.1% TMB (20x) in 100% Ethanol: Add 0.01 g of TMB (3,3’, 5,5’ tetramethyl benzidine, Sigma) in 10 ml of 100% ethanol (may be warmed to 37-40 °C to dissolve TMB) and store at 4 °C. 0.3% H2O2 (50x) in Distilled Water: Add 100 ul of H2O2 in 10 ml of distilled water and store at 4 °C. 0.2M Acetate Buffer-1% Sodium Nitroferricyanide (20x), pH3.3

Sodium acetate, trihydrate (MW 136.1) --------- 2.72 g

Sodium nitroferricyanide ------------------------ 1 g

Distilled water --------------------------------- 100 ml

Mix to dissolve and adjust pH to 3.3 using concentrated HCl and store at 4 °C.

0.01M Acetate Buffer - 0.05% Sodium Nitroferricyanide, pH 3.3:

0.2M Acetate buffer - 1% sodium nitroferricyanide --- 1 ml

Distilled water --------------------------------------------- 19 ml

Mix well and adjust pH if necessary.

Working Solution: Add 5 drops of 0.1% TMB to 5 ml of 0.01M acetate buffer – 0.05% sodium nitroferricyanide. Add 2 drops of 0.3% H2O2 and mix well.  Incubate sections for 20-30 minutes at room temperature.

Note: This is very sensitive solution and will produce more background staining than other peroxidase substrate solutions.