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Cytotoxicity - Science topic

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I used 0.5% Aq. AcOH solution to dissolve chitosan and derivative to evaluate their antimicrobial activity and performed anti-bacterial activity and antifungal activity using the broth dilution technique in 96-well micro-trays (NCCLS, 1993) and the antifungal screening method (NCCLS M27-A2 protocol, respectively. The cytotoxicity tests of prepared chitosan derivatives were assessed by MTT assay.
Now reviewer have following comment.
kindly help me in replying the comments of reviewers
comment of reviewer: if the derivates are still not soluble in water how are they be used in the kind of treatment proposed? What it's the implication of using acidic media in antimicrobial or cytotoxic treatment ?
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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
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I plan to study the cytotoxicity of metal compounds on 3T3 cells. However, I do not know which cell line to choose, i.e. Balb/3t3 or NIH/3t3 or perhaps Swiss 3T3 cell line. Which of these cell lines is easier to culture ? Do you have any experience ?
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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.
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Cytotoxicity of chitosan nanoparticles
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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,
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I have a molecule that changes the color of the media to yellow; when I was trying to use this for the LDH cytotoxicity assay, I had a lesser value than the test. As the compound was changing the color of the assay media, I used the compound alone control and performed the assay. I have an LDH control, and water control values are higher than the test. If I calculate the cytotoxicity, I will end up having negative value.
Is there any solution?
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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.
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Hello.
I would like to ask, how should I incorporate the cytotoxicity as a response in the factorial design, since cytotoxicity depends on concentration?
My only idea is to evaluate the IC50 for each experimental unit, but it seems like a horrendous amount of work.
Are there other approaches that would be more accessible?
Thank you in advance!
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Good luck Adam, I cannot think of any other way, research is often a horrendous amount of work.
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I have a compound that does not dissolve when I try to dissolve it with DMSO to determine its cytotoxic effect on cell lines (DMSO is not as toxic as 1%, but 0.5% is recommended).
I'm thinking to dissolve the compound with Hexane.
would you recommend hexane as a solvent?
What percentage of hexane toxicity is recommended?
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I investigated the cytotoxicity of several lipophilic compounds. I prepared the emulsion of Tween 80/PEG 600 (40/60 w/w) previously. When the lipophilic compound (once dissolved in DMSO) is emulsified in Tween80/PEG 600 and then diluted in the cell medium, no change in color of the cell medium was observed. The concentrations were: lipophilic compound 500 uM, DMSO 0.1%, Tween80 0.1% and PEG 600 0.15%.
To prepare the incubations with smaller concentrations, I first prepared the solvent, which I used for the dilution: DMSO alone was emulsified in Tween80/PEG 600 and then diluted in the cell medium. The concentrations remained the same. The color of the cell medium now changed (phenol red), indicating basic pH. Furthermore, the cytotoxicity assay showed that 50% of mortality is due to this solvent.
Can DMSO somehow disturb the emulsion and change pH? I can measure pH, but I need help finding out why adding only DMSO would change it.
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It can if the acid value of the Tween 80 and PEG 600 is on higher side
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direct cytotoxicity by crystal violet, could we use crystal violet to show the qualification of direct cytotoxicity of scaffold to cells?
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What are the criteria that I need
to consider while selecting secondary antibody for primary antibody for IHC experiment on mice brain?
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Dear Scientists of ResearchGate,
With your help and guidance, I was able to successfully grow L929 cells and use them for reliable cytotoxicity evaluation per USP <87>.
Now, I am updating the method to run as an XTT assay on a microplate reader. Does anyone have experience with that? The XTT assay kit is relatively easy to use and I'm in the process of determining the optimal cell density for each well in the microplate, but I can't seem to find much into on using the XTT assay for cell viability when adding a potentially cytotoxic compound.
Any feedback would be greatly appreciated. Thank you and best,
-Tim S
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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.
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I am trying to examine the cytotoxicity of an antioxidant agent on undifferentiated SH-SY5Y cells. In my previous try, all of the doses I tried gave results above 100% and all the wells were looking similar in color. I am not experienced in MTT assay, but I am thinking that I may change the doses. However, I am not sure how I should determine the new range. The doses I tried were 1 uM, 2 uM, 4 uM, 8 uM, 16 uM and 32 uM and I used 1 mM stock solution to dilute. I didn't change or skipped a step in the protocol but any ideas about the reason of failure is appreciated.
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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
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To investigate the effects of different drugs on the cancer cell, I did LDH assay and MTT assay. (p.s. The concentration of drugs was constant, I just looked at different drugs or their combination.) Briefly, I seeded cells in a 96-well plate, added drugs, and then put the plate in the incubator. After that, I took out the supernatant into a new 96-well plate to do LDH assay and the 96-well plate which was full of cells was used to do MTT assay. But, these results I got made me confused.
For example, the MTT value of one group was more than 100%, compared with the blank group, indicating cell proliferation. But the LDH %cytotoxicity of this group I mentioned before was about 3%, which meant some cells were killed. Ummm... I was confused about it. cell proliferation and cell death at the same time? The MTT results didn't correspond with the LDH assay.
Can anyone help me explain it? I thought there was something weird and I was wrong. I don't know why the MTT results didn't correspond with the LDH assay.
Btw,
LDH assay kit: CyQUANT™ LDH Cytotoxicity Assay Kit
And LDH %cytotoxicity is calculated according to the protocol.
MTT assay kit: CyQUANT™ MTT Cell Proliferation Assay Kit
MTT cell viability is calculated according to Christian Betzen's answer. https://www.researchgate.net/post/How_do_I_calculate_cell_viability_in_MTT_assay
Many thanks.
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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.
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What are some common and readily available options for inducing cytotoxicity in cultured adherent cells? I've been using hydrogen peroxide but discovered it interferes with a new assay and need an alternative. Thanks!
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I use DMSO 50%
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I am currently working on cell cultures and I'm treating various cell types with cytotoxic compounds. I read in an article about NDI ( nuclear division index) and I think it would be useful for interpreting my data. However, I can't find any information about NDI. Can someone help me ? Thanks!!!
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This is really helpful, thank you so much !!!
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Hi fellowScientists,
I am going use DMSO as a positive control in a cytotoxicity assay involving CACO-2 cells. I am looking for medium to high toxicity levels. Would 2% and 5% be acceptable concentrations? Thanks!
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Thanks, I was hoping for an answer from someone with a direct experience, reports vary.
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I am identifying cell types on Loupe Cell Browser using various cell surface makers. Are there any specific markers unique to NK cells that differentiate them from T-cells? Right now, I have filtered out the "NK cells" based on the fact that they don't express classic T-cell markers such as CD3E, CD3D, CD3G, CD8A, CD8B, TRAC; but do express genes required for cytotoxic functions such as GNLY, NKG7, etc.
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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.
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Hello,
I am conducting an experiment on testing the cytotoxicity of a drug on brain tumor cells. I am using Thermo Fishers alamarBlue reagent and measuring absorbance at 570nm/600nm to measure cytotoxicity. I have two questions:
-I have attached a photo of the equation that Thermo told me to use when calculating the % reduction. What does this equation mean?
-One of my supervisors is doing the same experiment but with different cells, she calculates percentage by merely averaging the blank values (Media + alamarBlue reagent) at 600nm and subtracting that from 570nm readings.
when it comes to calculating the IC50 value, which method is more accurate? because bot methods give totally different values.
I have attached the protocol from Thermo and the photo of the equation given by Thermo
Any help will be very much appreciated!
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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 = (E570,ox)Cox + (E570,red)Cred
∆A600 = (E600,ox)Cox + (E600,red)Cred
where C stands for concentration and E stands for extinction coefficient.
To solve for Cred, for example:
Cox = [∆A570 - (E570,red)Cred]/(E570,ox)
Cox = [∆A600 - (E600,red)Cred]/(E600,ox)
[∆A570 - (E570,red)Cred]/(E570,ox) = [∆A600 - (E600,red)Cred]/(E600,ox)
Cred = [∆A570(E600,ox) - ∆A600(E570,ox)]/[(E570,red)(E600,ox) - (E570,ox)(E600,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:
Cred = (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
Cred = (6.896)∆A570 - (4.739)∆A600.
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How can the in vitro cytotoxicity data of an extract be extrapolated for evaluation in an in vivo tumor model?
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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.
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The Peace on you
I have many question about papers of cancer and plants
1- can i calculate the IC50 from this table ( whatever cytotoxicity or vaiabilty) and does cytotoxicity =100-vaiabilty
2- what is the meaning and indication of selectivity index (SI)
3- Does there is a stander classification of ic50
like IC50=10-25 is potant
IC50=50-75 is moderate
IC50=>100 is non toxic
thannnnks in advance
thannnks a million
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1- can i calculate the IC50 from this table (whatever cytotoxicity or viability) and does cytotoxicity =100-vaiabilty.
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.
2- what is the meaning and indication of selectivity index (SI)
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,
SI= CC50 against normal cells/ IC50 against tumor cells.
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.
3- Does there is a standard classification of ic50
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.
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I did the Vero cell cultivation at different cell seeding density for toxicity effect study, and in the last two sets of MTT assays, cell died after MTT addition (even 10 minutes after, I just took a picture after 10 minutes and the cells were starting to detach from the surface, cell sheets detachment!).
In the first experiment, I had 3 different cell inoculation density in which I have the cell density range of low and high, but after MTT addition, they were all dead.
And at the second time, due to MTT reagent cytotoxic effect, instead of using 1:3 ratio of MTT solution, I used 1:6 ratio of MTT solution and the medium (I am using Cell Proliferation Kit II (XTT) from Roche)
I would appreciate any ideas or suggestions on what might be the reason for this.
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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
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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.
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I wonder how to measure cc50, someone told me to do the same way as finding IC50, now I finally got IC50 from Plaque Assay but still stuck with cc50 from MTT assays. If we do the same way to find IC50 then CC50 also using Cell viability to calculate same as IC50. Am I right ?
#cc50 #ic50 #cellviability #cellculture #verocell #MTT #Selectivityindex
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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.
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I did an LDH assay and have six concentrations of drugs I treated my cells with and when I put the LDH assay data in a graph, I see a normal distribution pattern. The 3rd concentration of the drug results in the highest cytotoxicity percentage but from the 4th concentration, the cytotoxicity decreases. How to decide on which concentration is the most suitable for my cells?
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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
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I want to check the cytotoxicity of my PHA sample, but it can only be dissolved in chloroform. I have checked its solubility in many solvents like dimethyl formamide, propanol, methanol, hexane, ethyl acetate, isobutyl methyl ketone, glutaraldehyde, formaldehyde, butyl acetate, ethylene glycol diacetate, DMSO. It is getting partially dissolved in 100% DMSO. and 50%butanol.
What solvent will be suitable for the cytotoxicity test.
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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.
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I checked the cytotoxicity of my composite in MG 63 cell lines. How could I interpret the cytotoxicity of my materials along with the cis platins IC50 values? I got 2.85 for my material and 37.5 for cisplatin. Is my material toxic?
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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.
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I have tested the cytotoxicity of a compound by MTT assay. The compound was quite active and gave appropriate growth inhibition. At IC50 dose, the compound was decreasing the gene expression of HIF1A but increasing the gene expression of VEGF as reported in literature that the HIF1A induces the VEGF expression.
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Thanks muhammad
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Hello. I am a beginner at using Graph Pad Prism software. I want to ask, is it possible for me to find the IC50 value for the cytotoxicity test if I only have the data for cell viability (%)?
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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
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Hi all, I have been doing MTT assay for a couple of months now but I have found it very difficult to get an IC50 through the readings of OD. On most occasions, I have witnessed an increase in OD as I increase my dose. My sample has reported cytotoxicity and the expected result should have been the reverse of what I'm getting now.
Is it merely troubleshooting? Or has anyone experienced this pattern as well?
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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.
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How does the passage number of a cell line affect experiment results including toxicity assays? Which characteristics of cells are changing as the passage number increases?
What is the most efficient or optimum passage number of cells (for example, for cancer cell lines such as HepG2, A549 etc. or for healthy cell lines like HEK293) for setting an experiment?
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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.
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My study is to test the effectiveness of nanoparticles on candida spp in cell-like environment. I need to culture epithelial cells and then add in the candida and nanoparticles. If I want to test on their gene expression, cytotoxicity level and others separately, how should I do?
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Mohsin Ali do you have such an expertise?
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I am going to use healthy liver cell line for the cytotoxicity experiments with nanomaterials. So, I am wondering that are there any differences between THLE-2 and THLE-3 cell line ?
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If silver nanoparticles show excellent antimicrobial activity then at the same time does this imply that their cytotoxic impact on mammalian cells will be more than other metallic nanoparticles? If that is so then for nano-enabled antimicrobial products, can silver be replaced by other metallic nanoparticles (in higher concentrations to compete with silver)?
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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
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What are the parameters for calculating cytotoxicity and is it possible to calculate them using DFT or any other computational methods. Please suggest relevant article also.
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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.
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Hello,
I'm working on the synthesis of pure curcumin nanoparticles using sol oil method described in this paper , but I'm facing two issues
1) nanoparticles are forming clumps in water and it is getting harder to resolve even with vigorous vortexing/bath sonication.
2) Not getting the desirable size in DLS particle analyzer. When we tried passing the nanoparticles down a 0.45 micron filter, we couldn't get any particles to pass through, suggesting heavy population of macroparticles.
What can be the possible ways to troubleshoot this issue?
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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.
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If nanoparicles (NPs) are prepared using the biocompartible polymers like HSA, BSA or PLGA of a certain drug(Say Doxorubicin) then the cytotoxicity exerted by the NPs could be higher than the native drug Doxorubicin in terms of their IC50 values? please provide some reference
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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.
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Hello, I am reading a paper saying their compound can lead to apoptosis of HepG2 cells; however, it came to me that is it possible that the cytotoxicity of the compound causes the apoptosis? Or what’s the difference between cell cytotoxicity and cell apoptosis?
Let’s say, If one compound is capable of leading to cancer cell apoptosis, how about its function on normal cells? Can it cause the apoptosis of the normal cell too?
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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.
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The synthesized compounds are found to be cytotoxic (<10), bind with DN but not cause any cell cycle arrest.
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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
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I would be grateful that somebody answers this question, I can't find the answer.
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Hi, in this paper you can see the aproximate amount for a good proliferation:
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I tested my drug, by MTT assay, on different cell lines (HeLa, Caco-2, HepG2, WI38, J774, Raw264.7) and I obtained different results in terms of cytotoxicity on these cell lines.
Can someone kindly explain the possible reasons for this difference in cytotoxicity on these cell lines?
Thanks in advance.
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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!
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Dear fellow researchers,
I wanted to know if I should use basal media for preparing working concentrations of paclitaxel, cisplatin and 5 fu. Are these drugs sensitive to FBS in media? How much percent FBS is allowed to be added along with drugs in basal media. Thanks in advance!
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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!
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Hi everyone.
I'm using MTT for cytotoxicity experiments with Vero cells. My question is about the calculations. I measured my samples at two wavelengths:
- 570 nm
- Reference: 630 nm
Please let me know if this is how we should do the calculations:
Should I correct each of my OD570 by subtracting the mean blank at 570 (Sample570 - MeanBlank570)
And correct each of my OD 630 by subtracting the mean blank at 630 (Sample 630 - MeanBlank630)
And then finally Adjust as follows: (Corrected570 - Corrected 630)
Is this how we use both Blank and Reference Wavelength in MTT calculations?
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Thank you very much for all your answers!! @Malcolm Nobre, that's very helpful.
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Hi researchers!
I want to run a mtt assay on PBMC to show that a cytotoxic drug in a leukemia, does not have any effect on normal cells I mean PBMC!
Is there any special protocol to isolate ,seed and treat Them?
does any one know how many cells can be seed in 96 well plate?
how about the drug concentration?IC 50 or less than IC50?which one is better?!
please help me,thank you
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great advice by Lorenz Waltl , but make sure to dilute it with PBS (after 30 sec) to make the soltuion isotonic again.
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I performed an MTT assay to see the effect of an inhibitor on AGS cells. I seeded them on Day 1, added the inhibitor on Day 2 and performed the MTT assay on Day 3 (left the inhibitor act for 24 HRS). I got a little weaker signal on the treated cells, compared to the control. How do I know if the inhibitor caused cytotoxicity or if it inhibited cell proliferation?
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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.
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Dear Scientists of Researchgate,
After a lot of trial and error, we managed to grow and maintain L929 fibroblasts in-house. We managed to grow a few monolayers that looked.. okay.
So far, what worked best for me was to plate 100,000-200,000 cells in 2mL of EMEM with 10% FBS for 24-48 hours, then replace the medium layer with pure EMEM (no Fetal bovine serum added). The cells differentiated relatively well.
Our team doesn't have any prior experience working with adherent mammalian cell lines, so I was wondering - does anyone have any tips on making sure L929 cells differentiate into fibroblasts with a higher percentage? And if anyone has experience working with these cells - could you please evaluate the quality of the monolayers I have attached pictures of? Any feedback would be greatly appreciated.
-Tim S
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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.
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I experience high cell death rates and I am looking for a buffer that can be used instead of the P3 buffer for the siRNA transfection of murine nTreg using the nuecleofactor Lonza system. Thanks!
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I don't think the P3 solution is toxic. Kindly optimize the nucleofection codes. That's the key. Consider using CA137.
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let me know details of the mechanism also
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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)
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First of all, I would like to thank those who share their knowledge.
We are conducting basic experiments to see cytotoxicity by treating cells with chemicals.
Chemicals ordered are soluble in DMSO or water in the same amount at 100 mM.
Is there a big difference between dissolving in DMSO or dissolving in water and treating the cells?
thank you.
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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.
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I want to bioconjugate a peptide with protected lysines with Dde onto gold nanoparticles so that it's only conjugated via the N-terminus. The idea is to deprotect the lysines after bioconjugation, and I would like to avoid using organic solvents such as DMF to do that because the gold nanoparticles are to be used in in vitro and in vivo studies afterwards. Eventhough the particles will be washed with water a few times afterwards I would like to avoid using any cytotoxic solvents if possible to minimize any risk of potential added cytotoxicity.
Is there any way to remove Dde groups in aqueous phase, if yes what are the conditions? If not is there any alternative or other protecting groups which I could use that you would recommend (peptide is synthesized through solid-phase synthesis)?
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See a similar question and the response:
Rikeshwer P. Dewangan
  • 27.65
  • School of Pharmaceutical Education and Research Jamia Hamdard New Delhi
How to remove Dde protection in solution phase reaction?
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  • Asked August 28, 2019
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
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related to Cytotoxicity and cell viability
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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.
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We are carrying out Cytotoxicity Study of a Pancreatic Cell line and we want to know the best protocol to use in the cell incubation.
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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.
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Upon checking individual ingredient toxicity for baby oil/body wash, found to be nontoxic. Later after formulation, the finished product was found to be toxic. We performed MTT assay to check cytotoxicity using L929 cells.Can some help in this regard.
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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?
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Hello,
I want to use Propofol, highly hydrophobic, in my cell culture media for human brain microvascular endothelial cells (HBMEC).
My stock propofol is 10 mg/ml (Mw: 178.271 g/mol)
The desired final concentration of propofol in my media that I need for exposing cells with, is 50 uM.
I'm wondering how I could dissolve propofol in DMSO and then in my media, by considering that the final concentration of DMSO should be 0.5% to avoid cytotoxicity.
I'd really appreciate it if you could help me with this!
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Ehsan Nozohouri , you're welcome.
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What is a good bio-adhesive with low cytotoxicity that could be used to subcutaneously implant an electronic device in mice, without having to suture the device to the tissue?
Any commercially available options? Dermabond and vetbond are not recommended for internal use.
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Check with these Fibrin Glue. These are the commercial names Tisseel, Vistaseal, Evicel, Reliseal Kit.
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Does anyone know what drug or method is suitable to remove the sulfate group from an intramembrane enzyme? Note that my goal is to work in vivo and I do not want cytotoxicity.
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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
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I am working on curcumin nanoparticles and their cytotoxicity towards the MCF-7 cell line. Whenever I performed MTT assay I get a high O.D while increasing curcumin dose concentration. I used 2micromolar to 60micromolar range of curcumin dose. Moreover, when I observed in microscope cell death has shown dose-dependent cytotoxicity. How do I have to consider my result? what should I have interpreted based on the absorbance data of MTT?
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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.
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Can I use plasma of the infected patient for doing Antibody dependent Cellular Cytotoxicity ?
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Thanks a lot for the answer @MalcolmNobre.
So, I need to purify specific antibodies against the antigen.
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Hi all,
I wanted to know if snap freezing the live tissue is helpful when one is aiming for paraffin embedding and sectioning (at room temp) later. Will the tissue gets damaged when it thaws?
My reason to go for the above approach is that putting the tissue directly in fixative which is generally toxic might alter the cell metabolism and lead to cytotoxic death.
I know the method depends on what you want to look at. It will be great if someone can suggest for various scenarios like structural/architectural studies, immuno staining etc
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Thanks all for your responses. I work with Drosophila by the way. not larger tissues like that of mice or human.
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Recently, I have been using the Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity Kit according to the Fluorescence Microplate Protocol.
Although it is not written in the protocol, I also prepared dead and live wells containing calcein-AM and ethidium homodimer-1 separately as it is needed in the calculation part.
But there is a part about the test results that I really couldn't figure out.
According to the results, the value I get is more than 100 when I sum up the percentages of the living cells and dead cells for the same well.
Commonly, I expect some cells to die, some cells to survive, but some cells to go into apoptosis.
When I reviewed the publications, I saw that most publications use the microscopy method. In fact, I read a few comments about the microplate protocol is not working properly in this assay.
I wonder is there anyone who has had the same problem before or if I am making a mistake while applying the protocol?
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Thank you for your valuable thoughts and time!!
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Dear Scientists of Research Gate,
Not too long ago I started working with an L929 cell line we need to keep in-house, and got the passaging conditions figured out. I even started cytotoxicity, but I seem to really struggle with storage.
Does anyone have any suggestions to a good cryopreservation protocol for these cells as well as a quality, reliable dewar intended for storage? I bought a cheap one, but I guess you get what you pay for - it leaks and kills my cells :)
Any feedback would be greatly appreciated.
Best regards,
-Tim M Seyidov
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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.
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I want to test drugs on different cerebral organoid slices (300µm+). Therefore, I want to characterize the cell viability of an organoid slice beforehand. Afterwards, the organoid slices shall be treated with different drugs and the cell viability shall be measured again. With this, I hope to measure the cytotoxicity.
So my questions are following:
Can I use a MTS assay to characterize a tissue such as an organoid slice?
Can I wash the sample thoroughly to do a MTS assay again with the same slice, or is there any problem I am not aware of?
Because its a tissue and not a small layer of cells, will there be any problem with the amount of cells for the MTS assay? I know it is usually only used for a very small number of cells, like in a 96-well plate.
Is there any other kind of assay, which would be better suited?
Thanks in advance!
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David Marks Had a similar question. Could you figure out the best way to quantify viability assay for organoids?
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While evaluating the cytotoxicity of a sample in cell line, what percentage of cell viability is considered inorder to conclude that the sample is toxic. Like if 80% of cell survives, is the sample toxic or not
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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
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Hi, I want to study the LD50 and cytotoxicity of an alcoholic , water extracts and the oil of plant in tissue culture and which lab animal is the suitable?
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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
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Does anyone know a small molecule toxin that comes as a two-component system? Or a real (highly) synergistic cytotoxic drug pair?
Thx
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There are tables in this article that may be of interest to you:
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Cytotoxicity study of anticancer compounds using Alamar blue on Cal27 cell lines
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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.
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I am planning an experiment to check the combination effect of 10 different chemicals for cytotoxicity. I know their individual effects and I would like to check their interaction effect or combination effect on cytotoxicity levels. Some compounds are pure biocides and some have low to moderate cytotoxicity. I came across fractional factorial design of experiments. I am not experienced with statistics so it is quite overwhelming to go through all the basics and calculations. What would be the most convenient way to carry out such an experiment? An expert's advice and help would be much appreciated.
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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.
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Hey All,
Our team wants to complete cytotoxicity per USP <87>, using the L929 cell line and I've been tasked with this. I've never worked with this cell line (or any animal cell lines for that matter) before, but I figured most of it out. We got all the right equipment, a liquid nitrogen dewar for long-term storage and I am capable of passaging cells well with high viability.
However, recently, I've noticed contamination in my cell line - mostly bacterial. The media changes its pH and turns pink, while some flasks turn visibly turbid as well.
For contaminated flasks, is there a way to de-contaminate cells? There are vialble L929 cells present, but adding 1% Antibiotic-Antimycotic doesn't seem to work too well.
Also, would anyone have any advice on preserving high viability of cells post-cryopreservation? I get my L929 from ATCC (they come at >80% viability), but my post-freezing viability average at ~58%.
Any help would be appreciated. Thank you!
-Tim M Seyidov
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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 -80oC).
To thaw a cryopreserved cell, prepare a 15c.c. tube filled with 9c.c. growth media, after take out the cryo-vial from -196oC or -80oC, vial should directly go into a prewarmed 37oC 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.
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So I would like to analyse two drugs (Drug1 has no antitumor effect, Drug2 is a used chemoterapeutic agent) and whether the Drug1 can antagonize the antitumor effect of Drug2. The tests were performed and the raw data seems to confirme this idea that's why I would like to validate it with the help of the CompuSyn software.
The data we have are the followings:
cell viability values of 2 concentrations of Drug1, cell viability values of 3 concentrations of Drug 2 and of the 6 combinations. When I enter these data set (I think correctly) in CompuSyn, the CI is not presented, an NaN (not a number) is shown.
It would be great if someone could help me out or give me some suggestions.
Thank you!
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If you were interested, also look at this link and the Compusyn software tutorial.
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Where are the MTT Cytotoxicity Evaluation Assay is available in India?, sample testing center for in vitro anti cancer activity on MFC-7 in india ???
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@ Jigar Soni sir
Please let me know pvt lab name
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Hello everyone
I'm doing MTT assay to measure cell viability of a cancer cell line after treating it with some drugs. Under the microscope, I could not see lots of cell death, but I could see that the wells with higher drug concentrations have less cells, their media is also more orange, the media of cells treated with less drug turned yellow. However, the MTT result is quite confusing because the wells with more drug/less cells have stronger purple color. The gradient of purple along the range of drug concentrations is opposite to what we expect from MTT assay. Has anyone had the same experience or do you have any opinions on this?
Thank you very much.
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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.
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I am currently working with U87MG cell line (glioblastoma). I need to perform MTT assay to check cytotoxicity of a compound. I have came across so many protocols of MTT assay. Which would be the best protocol for this cell line ?
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Please see this hopefully it will help.
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Dear People of Research Gate,
I am working on bringing a cytotoxicity assay by USP <87> into our lab. I've never worked with L929 cell line, but I think I have it mostly figured out. I was able to passage and properly cryopreserve cells while maintaining a ~90% viability.
I was curious if anyone could help me with running the oldschool USP 87 cytotoxicity protocol - we need to run that before we can integrate better colorimetric assays. I currently use Trypan blue.
Any advice or feedback would be greatly appreciated. Thank you!
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Dear Tim!
Please You look at the following links:
Cytotoxicity Growth Inhibition Test in L929 Mouse ... -
stratasys.com › Ultem-1010-Cytotox-Final-Report
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My work involves using pesticide formulations in vitro. However, even concentrations as low as 2.5% are proving to be highly cytotoxic and leading to cell-death. I have tried exposing the pesticide formulation for brief periods (~ 20 minutes) before changing the media to remove its traces (as recommended by the EPA protocol) but the cells do not survive (over 90% cell mortality rate)
It would be much appreciated if someone could recommend a protocol that would allow 50 % survivability .
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Dear Alexandr Chernov , thank you for the link to this paper.
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I am testing the cytotoxicity of certain antibiotics through MTT assay, On what basis should I select the proper starting concentration. And How to calculate the IC50 of the antibiotic.
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Hi,
I have been doing some experiments to reveal cytotoxic/antiproliferative effects of the new compounds in the cancer cell lines. After 24 hr incubation with compounds in Hep3B cells, I realized a specific structure on the wells with different compounds even my positive control (adriamycin), but not on control wells. So I am attaching an image of the wellhole shaped structure. Can you give an idea what this structure is or looks like? Thank you
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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)
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