Cytotoxicity - Science topic

Thank you for your response @Phil Geis. I checked chitosan and its derivative's antimicrobial and cytotoxicity on MCF-7 and L-132 by dissolving them in 0.5% acetic acid to make solution for biological evaluation. but reviewer asking me the implication of 0.5% acetic acid in such cancer treatment as my derivatives are showing higher cytotoxicity on L-132 and MCF-7. further we checked their effect on normal cell lines, but no derivatives and chitosan showed any significant effect on MCF-7 and L-132 cell line. Chitosan and derivatives had IC50 value of around 3000 ug/mL on normal cell line which indicates that chitosan and derivatives are non toxic to normal cell line but selectively toxic to MCF-7 and L-132.

Now could you please help me to give appropriate answer to reviewers comment.

Thanks and regards

Thank you very much for your detailed answer to my question. I was mainly hesitating between Balb/3T3 and NIH/3T3 and your answer helped me a lot in my choice. Thanks a lot.

Hi Asmaa,

I just read a new paper the other day that contained several separate assays which could be helpful with understanding cytotoxsisty of Chelate Coated Nanoparticles including Clonogenic assays, spheroidal assays and neurosphere assays.

Cheers,

If the compound's color is the only problem, then you can use the compound-alone control to measure the color and subtract that value from the cytotoxicity assay at each compound concentration in order to correct for the color.

However, if the compound is reacting chemically with something in the assay, then you should think carefully about whether that compound should be used, since its cytotoxicity may be non-specific. You could also consider using a different method to measure cytotoxicity that does not suffer from interference by the compound.

Good luck Adam, I cannot think of any other way, research is often a horrendous amount of work.

I would say no. Check out https://www.researchgate.net/post/Has-anyone-checked-the-effect-of-hexane-cytotoxicity-on-cell-lines-I-mean-as-a-control-and-what-was-the-result#:~:text=The%20cytotoxicity%20of%20hexane%20can%20be%20tested%20empirically%20using%20MTT,then%20go%20for%20cytotoxicity%20study. Might dimethylformamide (DMF) work? You'll need to assess (MTT, or Calcein-AM uptake, etc) solvent effects using your cell line and assay conditions.

It can if the acid value of the Tween 80 and PEG 600 is on higher side

What are the criteria that I need

to consider while selecting secondary antibody for primary antibody for IHC experiment on mice brain?

The XTT assay can not measure cytotoxicity but rather measures cell proliferation or a lack of cell proliferation. XTT/MTT assays are used for the measurement of cell proliferation in response to growth factors, cytokines and nutrients and can also used to measure the activity of growth inhibiting agents such as inhibitory antibodies i.e. a measure of cytostatsis. As such it does not provide an analysis of the percent or frequency of viable cells such as obtained with a trypan blue or PI analysis Nor does it provide an analysis of cytotoxicity such as would be measured with a 51Cr release assay.

You can measure a decrease in proliferation (cytostatsis) with this assay but it would be incorrect to label this a loss of viability or cytotoxicity. Hope this helps provide clarity.

Dear Irem,

Most likely the matter is in the nature of the substance that you tested. If your substance is an antioxidant, then it will reduce oxidative stress in cells, which means it will stimulate their survival, then what you got (survival rate is more than 100%). If you want to get a cytotoxic effect, you need to use oxidants.

Best regards,

Alexander

To be able to understand your results, you need to know what you are actually measuring - not relying on the commercial designation of the kits.

I use DMSO 50%

This is really helpful, thank you so much !!!

Thanks, I was hoping for an answer from someone with a direct experience, reports vary.

The main CD antigens on the surface of T cells are CD2, CD3, CD4/CD8. The surface markers of human NK cells are mainly identified by CD16 and CD56.

At present, CD3-, CD16+, and CD56+ are used as typical markers of NK cells. CD16 molecule is known as a low-affinity IgG Fc receptor. When the IgG class antibody specifically binds to the corresponding epitope on the surface of the target cell, it can bind to the FcR III on the surface of the NK cell through its Fc segment to exert a directional non-specific killing effect on the target cell, namely, antibody-dependent cell-mediated cytotoxicity (ADCC) of NK cells.

If you use the absorbance differences (the absorbance of the sample minus the absorbance of the control) at each wavelength, you would require a different equation to calculate the % reduced. The equation you showed earlier includes the control absorbances, so you should enter them into the equation.

As for the protocol used by your supervisor, I don't think any calculation of % reduced is involved. It simply uses the change in the absorbance measurement at 570 nm (minus the background from the culture medium, measured at 600 nm) to represent the degree of reduction. The extinction coefficient at 570 nm of reduced Alamar Blue is higher than that of oxidized Alamar Blue by almost 2-fold, so the more reduction occurs, the more the absorbance at 570 nm increases. To be honest, I don't understand why the background from the culture medium measured at 570 nm instead of 600 nm isn't subtracted in this method.

If you want to, it would actually be pretty easy to derive equations for the concentrations of oxidized and reduced Alamar Blue from absorbance measurements at 570 and 600 nm and the provided extinction coefficients if you first subtract the background from the culture medium at each wavelength (in other words the ∆absorbances.) It just involves a pair of equations and high school algebra for dealing with simultaneous equations.

∆A570 = (E_570,ox)C_ox + (E_570,red)C_red

∆A600 = (E_600,ox)C_ox + (E_600,red)C_red

where C stands for concentration and E stands for extinction coefficient.

To solve for C_red, for example_:

C_ox = [∆A570 - (E_570,red)C_red]/(E_570,ox)

C_ox = [∆A600 - (E_600,red)C_red]/(E_600,ox)

[∆A570 - (E_570,red)C_red]/(E_570,ox) = [∆A600 - (E_600,red)C_red]/(E_600,ox)

C_red = [∆A570(E_600,ox) - ∆A600(E_570,ox)]/[(E_570,red)(E_600,ox) - (E_570,ox)(E_600,red)]

The denominator is a constant, with the value 1.706 x 10¹⁰ in whatever units the given extinction coefficients are in, presumably M^-1cm^-1. Putting in all the constants gives:

C_red = (6.896 x 10^-6)∆A570 - (4.739 x 10^-6)∆A600

Convert from molar to micromolar to get rid of the 10^-6s, and

C_red = (6.896)∆A570 - (4.739)∆A600.

I don't think one can extrapolate in vitro cytotoxicity data into an in-vivo model. what you can do is "paw edema method' if your ultimate goal is selection of a dose.

Hello Dalia Youssef El-Berawey

I agree with Adam B Shapiro with regards to the table provided. It does not provide any clear information.

To calculate IC50 value you should plot a graph of cell viability (%) on Y-axis and log concentration of the drug on the X-axis. For this purpose, you need to run a cytotoxicity assay using mammalian cancer cells with increasing concentrations of the candidate drug. From the absorbance values, calculate the cell viability (%) as follows:

Cell viability (%)= average absorbance of test / average abs of control x 100

Then plot a graph of cell viability (%) on Y-axis and log concentration of the drug on the X-axis. Calculate the IC50 value from the graph.

An ideal drug should kill cancer cells, but not affect the normal cells. Therefore, the selective index (SI) plays an important role. You should not only evaluate the anti-cancer activity of the herbal drug on mammalian cancer cell lines, but also evaluate its cytotoxicity on normal cells.

SI is the ratio of the toxic concentration of the candidate compound against its effective bioactive concentration.

Known as the tumor selective index,

where CC50 is the concentration of the candidate drug to cause death to 50% of the normal cells while IC50 is the half-maximal inhibitory concentration which is a measure of the candidate drug in inhibiting a biological function.

In an ideal situation, the IC50 value should be as low as possible while the CC50 value should be as high as possible which means that the drug is highly effective against cancer cells with minimal effect on normal cells. So, SI value gives you an idea about the selectivity of the drug. Greater SI value means your drug is more selective in killing cancer cells.

As mentioned by Adam B Shapiro there is no such thing as standard classification of IC50 value. However, in literature there has been a mention of it namely,

IC50 can be categorized as high (IC50 value below 1uM),

moderate (IC50 between 1uM and 10uM), and

low (IC50 above 10uM).

Low IC 50 value means that the drug is effective at low concentrations, and thus will show lower systemic toxicity when administered to the patient.

Best.

Hi, I'm finishing up my PhD in a venom analysis lab. I do massive amounts of toxicity, cytotoxicity, toxic mechanisms, differential and selective toxicity, and use everything from counting cells by eye, by trypan blue stain using a Countess, by various flow cytometric assays, and my PI has been at the forefront of venom analysis for decades. When I am contracted by pharmaceutical companies trying to develop a drug they prefer MTT assays as their initial "cytotoxic or not" and "approximate EC50 dose". Even though it's indirect it is far easier to run multiple replicates of multiple doses even when you have little material (such as an isolated scorpion venom protein... it takes a lot of, potentially hazardous, work to get a few micrograms sometimes). So I am intimately familiar with MTT and (if you haven't looked into this, I recommend it) MTS.

As for why you are seeing cells die after the addition of even small amounts of MTT it is because MTT is extremely cytotoxic to any cell that produces NADH (so... cells that do cellular respiration) is that MTT kills cells. "Intracellular metabolism of MTT has been shown to gradually cause mitochondrial injury, disturbance of normal cell metabolism, and finally cell apoptosis [4,41]. Time-dependent loss of membrane integrity as a result of formazan exocytosis has also been proposed as a mechanism of cell death following MTT incubation" from

I don't think you pipetted too hard. You need to titurate cells well following MTT treatment to get a good result and you need to solubilize the cells with DMSO or another detergent. Formazan (reduced MTT) forms insoluble crystals, you can see these after treatment with MTT, these have been shown to build up in multiple organelles including mitochondria, ribosomes, nucleus, and not only interfere with cellular operations but can pierce and lyse cellular membranes in a similar manner to any crystal forming within a cell (such as ice). Even in extremely low doses I think you would witness apoptosis induction in healthy cells, and the loss of adhesion due to loss of membrane integrity and effective protein synthesis and the consumption of the NADH necessary to maintain vaiability. MTT is not an assay that is meant to result in intact and definitely not living cells. It is essentially quantifying the amount of reducing agent present (which is typically correlated to cell number, but it varies for every cell type and every treatment and you should always have an identical treatment ladder with varying cell numbers ranging from zero to double what your treatment wells will be seeded with). The nice part of this is that I have found that incubating my cells with MTT overnight results in a far more consistent result, and the use of a ladder as described below demonstrates whether the cell numbers used, incubation time, MTT dose, and carrier provided a relationship between seeded number of cells and optical absorbance that has (for my purposes) an R2 value of over .9 for either logarithmic, asymptotic, or linear best fit lines. It's also acceptable, in my opinion, to exclude the double population and lowest population if these are outside a clearly linear, with high R2 value, relationship for all other cell numbers and absorbances. However, this also means that you should not be including any treatments where the results were outside your new maximum and minimum (the lowest viability would be the lowest population you included in you best fit defining data from the ladder). This is also typically only useful in the case of analyzing cytotoxins by dose and trying to find the dose that results in an equal absorbance as if you simply seeded the wells with half the cells, since if you have 100%, 50%, 25% and 12.5 % but 200% and 6.125% were not within the linear range it doesn't mean you need to throw out all your data... most treatments will be somewhere between 100% and 12.5% relative viability anyway... assuming there was some time that the cells were alive and produced some NADH then even only 1 hour should have been eough time for the 100% population to produce as much as a population 1/24th it's size if incubated for 1 day.

Anyway, I just wanted to elaborate a bit and explain why I am of the opinion that trying to keep your cells alive after exposure to MTT is an unlikely task. MTS is water soluble and does not form crystals and it may be possible to find a dose that is both observable colorimetrically and may not be completely wiping out the populations... but for MTT I don't think you would get surviving cells if you've added a dose that is useful for quantifying things at any level.

. If you don't use a ladder (every single plate i use for an MTT assay has 4 columns devoted to a ladder, the first containing double the cell population as my treatment wells, the second identical, the third half my treatment, then a quarter, then an eighth, and finally a media only and a carrier +media only, this ladder should result (with 4 reps at each population) in relatively consistent results (they should be seeded at the same time as the rest of your wells and treated identically to your control wells, same volume of MTT, same incubation, read at the same time... it's on the same plate so this shouldn't be difficult to make the conditions close to identical except for position on the plate... so remember that corners and edges evaporate faster and will often seem slightly darker and more absorbant as the same quantity of f

Hello Lamis Saleh

If you have a compound showing anti-cancer activity, then you will have to perform cytotoxicity assay such as MTT to check for its potency. Potency is an expression of the activity of a compound in terms of the concentration or amount of the compound required to produce a defined effect. When you perform the MTT assay on mammalian cells preferably cancer cell line using an increasing concentration of the candidate drug you will see an effect i.e., with increasing concentration of the candidate drug there be a corresponding decrease in cell viability.

Plot the graph of drug concentration on X-axis and cell viability(%) on Y-axis. Calculate the IC50 value from the graph. IC50 value is the half-maximal inhibitory concentration which is the most widely used and informative measure of a drug's efficacy. It indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of potency of the drug. Lower the IC50 value, more potent will be your compound.

Having a compound showing anti-cancer activity does not mean that it has high cytotoxicity. You need to find its potency by performing the cytotoxicity assay.

Best.

CC50 is done when you have established a significant activity as against a particular micro-organism. The cytotoxicity assay is done using the same concentrations used during the screening (serial dilutions of 6 to 8 different concentrations) on sensitive cell (Vero cells or monkey kidney epithelial cells). The cells viability can be check by MTT/Formazan assay and the data obtained analyzed using a software and the CC50 value obtained. You can then calculate the selectivity index (SI) which is a ratio CC50:IC50. The value obtained will tell you how safe your product is. When your SI value is greater than 1, it's safe but the higher the magnitude of SI the safer and more important the product becomes.

Dear Fa Ro ,

There are different approaches depending on your design (do you want it to kill your cells? What will you use this concentration for?). The most common one is IC50 where you end up having both death and living cells.

Here are some common approaches:

1. IC approach: You pick a concentration which kills a certain percentage of your population (IC%). Most common one is IC50 (concentration which kills half of your cells) but you can go for IC25 or IC75 as well.

If you do not have the exact IC% value, you may pick the closest concentration or you may draw a line or a curve in your graph and then calculate from the slope (equation).

2. Lowest effective concentration. You pick the lowest concentration which kills your cells (viability becomes lower than 100% for the first time).

3. Highest ineffective concentration. You pick the highest concentration which does not kill your cells (viability is still 100%). This is usually when you want all of your cells alive (combination studies).

Cheers.

Burak

We have applied a test for bacterial endotoxins that may be associated with PHA samples by analyzing powdered PHA samples prepared by cryogrinding under liquid nitrogen which avoids using solvent which may affect the assay.

hello Athul Ravi

If both of your results are in same units (µM or µg/mL), it is obvious that your composite is much more cytotoxic than cisplatin because your composite ic50 is about 13 times more than cisplatin.

Thanks muhammad

Dear Farisya,

Graphpad Prism is a very useful tool to calculate the IC50 values of the cytotoxicity, enzyme inhibition, ELISA based assays. It also provides good quality of figures for potential publications.

This video will help you to manage your data for the calculation.

Bests

Adem

Hello Trinayan Barthakur

Sometimes, there could be chemical interference of the test compound. You could confirm this by measuring absorbance values from control wells without cells incubated with culture medium containing MTT and various concentrations of the test compound.

Best.

Passage number as the number of times cells have been transferred from vessel-to-vessel, which affects a cell line's characteristics over time as the passage number increases their phenotype and genotype can change. Hepg2- Better to do within 16 passage, A549- Better to do within 20 or 30 passage, normal Hek293- Better to do within 20 passage.

Mohsin Ali do you have such an expertise?

Thanks Malcolm Nobre

There are cells that show cytotoxicity from Silver [sperm cells for example] However, Ag is a broad-spectrum antimicrobial while other metal particles, Zn ,Cu have limited targeted appeal, I am attaching a publication on our Ag particles and the mode of action. I have not investigated cytotoxicity effects with other metal particles .I woudl be interested in doing that

The short answer is no.

This said, one of the reasons is that the mechanisms of cytotoxicity are very different. For example, it is well known that Pt drugs, such as Pt(NH₃)₂Cl₂, are cytotoxic because of a mechanism of direct interaction with DNA. This notwithstanding in other cases cytotoxicity is due to redox activity. For example, a few gold compounds owe their cytotoxicity as antimicrobial activity to their Au(I)/Au(0) or Au(III)/Au(I) equilibria that renders them moderately oxidant. As far as antimicrobial agents are regarded I and my coworkers reported such an explanation in this past work:

As far as cytotoxicity is regarded, something similar holds. For example here:

we reported on a relationship between cytotoxicity (IC50) against the A2780 ovarian carcinoma cell line and the Kohn-Sham LUMO eigenvalues for a library of several Au(I) and Au(III) complexes.

In summary, if it may be true that sometimes a model mechanism (DNA intercalation, redox activity) can at least partially account for cytotoxicity , the opposite is absolutely not.

So (this was the long answer) there is no a priori method or technique to calculate the cytotoxicity of a compound.

You need to add capping agents. You can go for PVP or natural edible polymer chitosan. Capping agents will prevent nanoparticles to get aggregate. You can also optimize size of AgNPs by response surface methodology.

I think it is no sense to compare native drug and nanoparticles one. Just compare native drug and prescription. It is certainly that the nanoparticles exhibit a stronger toxicity.

Hello Li Qingyun,

Apoptosis is a form of programmed cell death that has important roles in development, aging, and disease. Apoptosis is initiated by a tightly regulated signaling cascade that results in caspase activation. Several features characterize apoptosis, including cell shrinkage, membrane blebbing, chromosome condensation, nuclear fragmentation, DNA laddering, and the eventual engulfment of the cell by phagosomes. Two major pathways are operated during apoptosis. The intrinsic cell death pathway is governed by the Bcl-2 family of proteins, which regulate commitment to cell death through the mitochondria while Activation of the extrinsic cell death pathway occurs following the binding on the cell surface of “death receptors” to their corresponding ligands such as Fas, TNFR1, or TRAIL.

On the other hand, cytotoxicity is the degree to which a substance can cause damage to a cell. A substance or process that causes cell damage or death is referred to as cytotoxic. Cells that are exposed to cytotoxic compounds may undergo necrosis (uncontrolled cell death), apoptosis (programmed cell death), autophagy, or stop actively growing and dividing to decrease cell proliferation.

Drug induced mild insult could induced apoptosis, while high level of insult or sustained high level of insult may induced cytotoxicity. The drug-mediated cytotoxicity may operate several death pathways including apoptosis, necroptosis, necrosis etc depending on the level of insult generated by the experimental drugs.

Best

The answer is "yes".

Although its very difficult to explain herewith the the effects of anticancer drugs on various cellular cellular physiology including cell cycle arrest and/or cell death pathways under in vitro culture conditions specially when you are trying to synthesize and develop anticancer drugs in Chemistry Lab.

It seems that you are synthesizing drugs keeping in mind that could be used as "anticancer drug". And the synthesized compounds in you hand is found to be cytotoxic (<10). Am I right? If yes, at this juncture, its very premature to say that it is anticancer drug, until you carry out a detailed study and establish it.

There are several anticancer drugs that interact with cellular DNA and exert cytotoxic effect under in vitro culture conditions. Indeed, the identification of DNA binding compounds can be accomplished by observing altered migration of supercoiled, covalently closed, circular DNA (scccDNA) in agarose gels. However, one simple procedure is to measure the ability of a molecule to bind to DNA by size–exclusion high pressure liquid chromatography (HPLC) measurements on a DNA–compound mixture.

You may have to understand the basics of cellular physiology in greater detail for analyzing the extent of drug insult that could induce cell cycle arrest, initiation of apoptotic cell death, necroptosis, autophagy and necrosis. If the drug insult is high and cell is unable to tolerate, it can induce toxicity quickly without affecting the cell cycle machinery. However, if the insult is mild then it may induce generation of ROS that finally results in cellular stress under in vitro culture conditions.

Increased level of ROS cause cell cycle arrest and thereby apoptosis. Under this condition, if the stress is mild, cell gets enough time to program for the death so called "programmed cell death". However, if the stress level is very high then cells do not get time to program (decide) and unable to tolerate the adverse conditions, then quickly undergo necroptosis or necrosis and cause cytotoxicity.

Indeed, cell cycle arrest and apoptosis could be interlinked.

Best wishes

Hi, in this paper you can see the aproximate amount for a good proliferation:

Results of MTT are dependent on many factors as mentioned by other above.

In simple words, MTT works on how energy active the cells are i.e., number of mitochondria present in the cells. More mitochondria mean more conversion of the dye (3,4,5-dimethyl.......tetrazolium bromide to formazan), results in higher color and hence different reading. It is known that metabolically active cells like heart cells have higher mitochondrial content as compared to HEK or 293 cells. Further in your list, you have mouse macrophage line (Raw264) that would be different from other human cell lines. Macrophages are relatively more active then say HepG2 cells. So the reason of your variability with the same drug is highly dependent on the mitochondria content in these cell lines.

Secondly, cell densities would also affect MTT results. You may have seeded for instance, 10K cells for each cell line but each cell line has different growth kinetics, so one cell line would be at 11K whereas another at 15K at the time of the assay, this would also alter the readouts. This is one of the reasons now days MTT needs to be supported by other non-metabolic cytotoxicity evaluation methods.

Best of luck!

In some previous work I did with paclitaxel, we dissolved it in DMSO (5 mg/mL). Dissolving drugs directly into media is the most ideal method as you have less controls to deal with. However, if you choose to use a solvent like DMSO or ethanol which provide better solubility for many drugs, just be sure to include a control that contains the same percentage of DMSO/ethanol dissolved directly into media alone (without drug) to account for any background noise when running plates, etc. Yes, you will eventually have to dilute your working stock solution in media in order to actually treat the cells. Hope that clarifies things. All the best!

Thank you very much for all your answers!! @Malcolm Nobre, that's very helpful.

great advice by Lorenz Waltl , but make sure to dilute it with PBS (after 30 sec) to make the soltuion isotonic again.

Hi Osmar,

A possibility is determine cell viability by means of the trypan blue exclusion test, in order to compare the number of viable cells after treatment comparing with the number of viable cells in the untreated cells.

To clear your curiosity, let me tell you under culture conditions, healthy cells will show all physiology including adhesion to the substratum (for adherence cells/cell lines), elongation, spreading and division etc.

If the cells are starved (if cultured in serum free medium) or exposed to sub-optimal conditions that can be easily observed by reduced attachment, elongation, spreading and focal adhesion points.

On order to minimize the exposure of insult/stress, cells under in vitro conditions show various morphological changes and try to reduce the surface area. Hence, the cell adhesion, elongation and spreading properties of the healthy cells are adversely affected.

If the level of insult/stress is more then you can also see the cytoplasmic granulation, cell shrinkage, membrane blabbing, and even cytoplasmic fragmentation in some of the cultured cells. Actually these the morphological features of apoptosis in stressed cells.

You can see some of the cells in the image are showing good attachment, increased cytoplasmic area and spreading with number of focal points, indications of healthy cells.

Best

Dear Nadja Nicola Ilkenhans ,

I don't think the P3 solution is toxic. Kindly optimize the nucleofection codes. That's the key. Consider using CA137.

Mutagenic NOT means cell increase. Mutations are caused by faulty DNA replication (especially meiosis) or faulty repair of DNA after damage (such as exposure to radiation or carcinogens)

Hello Jaeseung Son

Yes, there is a difference between dissolving chemicals in DMSO and water. DMSO is cytotoxic to cells. A high concentration of DMSO should never be used for cell culture. Most cell lines can tolerate from 0.1%- 0.5% DMSO as the final concentration in culture. However, primary cell cultures are far more sensitive. So, for primary cells one can use a final concentration well below 0.1%.

So, if you have an option to choose between DMSO and water, you should dissolve the chemicals in sterile distilled water and treat the cells.

Best.

See a similar question and the response:

Rikeshwer P. Dewangan

How to remove Dde protection in solution phase reaction?

Question

I have a reaction in which two Dde protection, which i want to remove in solution phase reaction. I have done the deprotection of this group in solid phase peptide synthesis and its very easy to remove by using of 2% hydrazine in DMF. What kind of the solvent should i use to perform this reaction and how to work the reaction further.

Carlo Pifferi added an answer

September 19, 2019

I would perform the Dde de-protection in solution exactly as you did (2% hydrazine in DMF) in the solid phase. This reaction is really efficient (I would say in 10 minutes at 500mM conc. is already finished). Since you are working in solution phase, I suppose that you would like to avoid to use DMF. In that case, depending on your sequence and the degree of protection of your residues, I would try with MeOH, DCM, or acetonitrile. Keep in mind that, since the deprotection mechanism involve a nucleophilic attack, if you have neighboring amino groups (for example, a free Lys which is close to your Dde-protected Lys) Dde might migrate.

Check this out --> 10.1111/j.1399-3011.1998.tb00630.x

Best regards

Hello Manish Gaur

Nanomaterials can interfere in assays through quenching of transmitted light or fluorescence. Therefore, careful consideration must be given to fluorimetric/colorimetric in vitro toxicological assessments of optically/chemically active nanomaterials in order to relieve any potential artifacts due to the nanomaterials themselves.

Please refer to the article below for more information.

I would recommend the use of MTT assay. Please refer to the review article below.

Best.

Hello Chika Unegbu

Shakers are not required. The incubator in which the cells are incubated maintains optimal temperature, humidity, and other conditions such as the CO2 and oxygen content of the atmosphere inside. For the cytotoxic assay, just add the candidate drug, place the plate on the shaker for 3-5 mins and then incubate the plate in the incubator as per the required time.

Drug molecules get into the cells either by sliding in through the membrane on their own (passive diffusion), or they're taken up by pores and proteins present on the cell membrane to get into the cells (active transport).

Best.

MTT assay using L929 cells@10 K is used., allowed to attach for 24 hrs. Sample preparation. 0.1 % weighed or pipetted and dissolved in DMEM medium with FBS and sonicated for 20 hz for 5 min. Then 100 ul loaded and treated for 24 hrs, then media is removed, washed twice using PBS. MTT reagent was added and then incubated for 3 hrs and so on.

Is this procedure is correct, mainly sample preparation?

Ehsan Nozohouri , you're welcome.

Check with these Fibrin Glue. These are the commercial names Tisseel, Vistaseal, Evicel, Reliseal Kit.

Kashif Ameer

Do you think it causes toxicity for the whole cell? Do you have another suggestion for inactivating the enzyme? I do not want to use the usual medicines.

Thank you very much for your response

Nilam Parmar phytocompounds such as curcumin have maximum absorbance peak at a wavelength ~425 nm, may be curcumin nanoparticle has absorbance wavelength that is interfering with MTT reading wavelength. Try analysing absorption spectra of your sample first. Try 650nm for MTT assay, if that also do not work find alternative cytotoxicity assay or try XTT assay.

Thanks a lot for the answer @MalcolmNobre.

So, I need to purify specific antibodies against the antigen.

Thanks all for your responses. I work with Drosophila by the way. not larger tissues like that of mice or human.

Hello Shian-Ren Lin

Thank you for your valuable thoughts and time!!

Dear Tim!

Please You look at the protocol:

Molecular mechanisms of cell cryopreservation with polyampholytes studied by solid-state NMR

Cell culture

L929 (mice fibroblast cell line, American Type Culture Collection, Manassas, VA, USA) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS). Cell culturing was carried out at 37 °C under 5% CO2 in a humidified atmosphere. When the cells reached 80% confluence, they were removed using 0.25% (w/v) trypsin containing 0.02% (w/v) EDTA in PBS (−) and seeded on a new tissue culture plate for subculturing.

Cryopreservation protocol

The cryopreservation solutions were prepared as follows: COOH-PLL was dissolved in DMEM without FBS at a concentration of 7.5% (w/w), and the pH was adjusted to 7.4 using 5 M HCl or NaOH. The osmotic pressure was measured using an osmometer (Osmometer 5520; Wescor, Inc. UT, USA) and was adjusted to ~600 mOsm for cryopreservation solutions of PLL derivatives using a solution of 10% NaCl. The cells were counted and re-suspended in 1 mL volumes of saline solutions of COOH-PLL, PEG, BSA, and DMSO with various concentrations at a density of 1 × 106 cells/mL in 1.9 mL cryovials (Nalgene, Rochester, NY) and stored in a −80 °C freezer after cooling at a rate of −1 °C/min in a controlled freezing container (Mr. Frosty, Nalgene) over 1 w.

Cell viability assay

Individual vials were thawed in a water bath at 37 °C with gentle shaking, after which the thawed cells were diluted in DMEM. Following centrifugation, the supernatant was removed, and the cells were re-suspended in DMEM (5 mL). To determine the cell survival, the medium was collected, and all cells were stained with trypan blue and counted using a hemocytometer immediately after thawing. The reported values are the ratios of living cells to the total number of cells.

David Marks Had a similar question. Could you figure out the best way to quantify viability assay for organoids?

Calculation of LC50 value by MTT assay seems to be the best way.we can take two concentrations below it and two above in it for some selected time of exposure

Hi dear Dr Luma, Thanks for your excellent question, I attached some references and also refering the same question and page here in Research gate, Hope they could be useful for you, otherwise please tell me to present another

1- https://www.researchgate.net/post/What-LD50-in-a-cellular-toxicity-assay-of-a-plant-extract-should-be-considered-as-low-toxicity

2-

There are tables in this article that may be of interest to you:

Hello,

Janhavi Bhadwalkar, The Alamar Blue assay may not be affected by dead cells. The Alamar Blue Cell Viability Assay reagent assesses the proliferation of mammalian cell lines, bacteria, and fungi in a quantitative manner. The dye contains an oxidation-reduction (REDOX) indicator that fluoresces and changes color as the growth media is chemically reduced due to cell growth. Viable cells convert resazurin to resorufin on a continuous basis, yielding a quantitative measure of viability.

The oxidized version of the Alamar Blue (resazurin) dye is blue and non-fluorescent. Growing cells produce a chemical decrease of the Alamar Blue dye from non-fluorescent blue to red fluorescence in the Alamar Blue assay. A reducing environment (fluorescent, red) is maintained by the ongoing development of viable cells, whereas an oxidizing environment (non-fluorescent, blue) is maintained by the inhibition of growth, which can be detected with a fluorescence or absorbance detector. If your drug works successfully on the cell line (Cal27) you're using, you'll see a concentration gradient dependant fluorescence absorbance reading. As a result, dead cells could not cause any interference in the assay.

Best wishes.

Hi Siddharth;

for the factor 0.05, this means that you have an assurance that your model therm is significantly different from zero to 95%. I think it's good to keep 0.05, to stay within the usual norm. And for the fact that I = ABC can be mixed with its alias, you have to check the resolution of your model, to detect with which therm your aliases are mixed.

Our lab rarely have encounter cell contamination, for what I remember, Normocin from InvivoGen have been advised by lab co-worker for prevention. In case of contamination, after disperse L929 with trypsin-EDTA (ATCC recommended 0.25%, but I think 0.05% should work), we will first use same amount of growth medium to attenuate trypsin's activity, then add PBS to wash off or dilute any bacteria in medium (up to 3 times), then provide a fresh prepared antibiotic (you may also switch antibiotic to deal with resistant strain) (1% penicillin/streptomycin or 0.2% Normocin) containing growth medium and observe for another 2 days. The reason is maybe the medium been through too many freeze-thawing cycle, the antibiotic can loose its inhibition ability, there will be a chance to inhibit bacterial growth by adding a fresh one.

For L929 cryopreservation, ATCC recommends 5% DMSO, and a single cryo. vial we do not store more then 1x10⁶ cells. After subculture, remove the sup. then apply 1ml medium-DMSO mixture and freeze the cell as soon as possible (we use cell freezing container and directly into -80^oC).

To thaw a cryopreserved cell, prepare a 15c.c. tube filled with 9c.c. growth media, after take out the cryo-vial from -196^oC or -80^oC, vial should directly go into a prewarmed 37^oC water-bath, do not wait until the ice melt completely, but left a small piece of ice floating, just before adding into media containing tube it should be totally melted, this can slightly increase cell viability.

Same procedure have always bring >85% viability in our cell lines.

Best of luck.

If you were interested, also look at this link and the Compusyn software tutorial.

@ Jigar Soni sir

Please let me know pvt lab name

In your case I think that during your experiment you should include the interference wells ( cells+media without MTT) to evaluate the influence of dyes. Also you should include the blank at different concentration ( drugs without MTT) to see if your drugs alone absorb because most of color samples reacted differently with dyes.

Please see this hopefully it will help.

Dear Tim!

Please You look at the following links:

Cytotoxicity Growth Inhibition Test in L929 Mouse ... -

stratasys.com › Ultem-1010-Cytotox-Final-Report

Dear Alexandr Chernov , thank you for the link to this paper.

Please see our paper for more info

Mutant p53s and chromosome 19 microRNA cluster overexpression regulate cancer testis antigen expression and cellular transformation in hepatocellular carcinoma | Scientific Reports (nature.com)