Questions related to Cytotoxicity
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 ?
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 ?
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?
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!
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?
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.
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,
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.
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.
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
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!
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!!!
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.
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!
How can the in vitro cytotoxicity data of an extract be extrapolated for evaluation in an in vivo tumor model?
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
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.
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
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?
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.
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?
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.
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 (%)?
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?
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?
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?
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 ?
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)?
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.
I'm working on the synthesis of pure curcumin nanoparticles using sol oil method described in this paper
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?
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
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?
The synthesized compounds are found to be cytotoxic (<10), bind with DN but not cause any cell cycle arrest.
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.
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!
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?
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
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?
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.
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!
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?
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)?
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.
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!
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.
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.
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?
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
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?
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.
-Tim M Seyidov
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!
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
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?
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.
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
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.
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 ???
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.
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 ?
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!
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 .
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.
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