- Nazlı Ezgi Özkan added an answer:Can I use MTT protocol tfor short time incubation?
How is the best protocol to measure viability with MTT reagent when I need to incubate the cells with some drug for 30 minutes?
Can I wash the cells treated with this drug and after incubate with MTT reagent?
Thanks a lot!
30 min incubation-->wash-->WST1 (3 h), works for my experiments !Following
- Carlos Araújo Queiroz added an answer:How can I control Ph in the bioreactor?
CHo cell was used to produce recombinant protein. In the shake flask the cell viability is very good. But in the 5L bioreactor the CHo cell viability will decrease quickly. Maybe the pH control will influence the cell viability.Could any one give me some advice?
It is generally much simpler to efficiently control the pH in a bioreactor than the concentration of dissolved oxygen (DO), which can have a critical effect for aerobic fermentations. I have studied adaptive control of DO and the related energetic optimization of aerobic fermenters taking air flow and agitation speed as manipulated variables. The parameters from the usual KLa correlation were estimated using sinusoidal excitation of air flow and agitation speed through the recursive least squares algorithm with forgetting factor. The original adaptive control algorithm used for DO compared favourably to PID when tested by numerical simulation. Cf. C.M.G.A. Queirós, "Controlo do Oxigénio Dissolvido em Fermentadores para Minimização de Energia Consumida", MSc Thesis, Instituto Superior Técnico, Universidade Técnica de Lisboa (Lisbon), 1997 (in Portuguese).
- Steingrimur Stefansson added an answer:Does anybody know of a live cell fluorecescent staining reagent that is fixable by fomaldehyde and retained?
Hi all, I'm looking for a fluorescent live cell staining reagent that can be fixed in situ by formaldehyde and not be easily washed out. Does anyone know of such a reagent?
Hi Philippe, great suggestion!
I have been using the live cell stain CellTracker™ Blue CMAC (7-amino-4-chloromethylcoumarin) to identify live CTCs in whole blood samples of cancer kept in our preservation solution (HemSol) at RT for over 5 days.
Some of the CTCs are staining weakly with CMAC. This could be due to the CTCs being apoptotic.Following
- Ravi Kant Upadhyay added an answer:Can I use neutral red as vital stain for bioassay of marine copepods?
Intra vitam staining with neutral red dye vividly stains live copepods, providing a rapid coloration to the tissues.vital stains such as SYTOX green is also used to bioassay marine phytoplanktons.DAPI staining is also used.Following
- Ishan Goswami added an answer:How do you properly use alamar blue for a continuous monitoring over a period of time?
I would like to monitor cell metabolism over a period of several days (possibly beyond a week). Which of the two methods is correct? Or are both okay to do?
a. Make separate wells for each end-point time (e.g. after 11d, after 3d, etc.)?
b. Use the same well at each time point?
Thank you very much for clarifying
Thanks both. Marina, that paper was really good! Appreciate your technical input on this.Following
- Ishan Goswami added an answer:How does CyQuant Direct compare to CyQuant NF? Could somebody please share their experiences?
I am contemplating using a CyQuant line of dyes from Life Technologies (LT). But there are two types which are confusing me: CyQuant Direct and CyQuant NF. From their description, the only difference seems to be that the Direct needs the removal of a supernatant in a well-plate format. I was further confused by a tech rep from LT who told me CyQuant just gives you a total count of live and dead cells, contrary to what I read in the description.
Hi Mannan, Thanks for the response. Does it stain dead cells? I am little worried about their propaganda. There is a similar staining agent NucBlue which a colleague of mine is having trouble with since it also staining the dead cells.Following
- Renato Konrath added an answer:Why tannins give a false positive in the biocompatibility test in mangosteen skin and chlorhexidine gluconate 0,2% against BHK-21 fibroblasts?
My research using the MTT assay. The results of my research show false positive results. I want to ask, what makes Tannins give a false positive result on this test? And what is the ideal requirement to obtain positive results in the MTT assay test? Thank you.
Monika, I'm not very familiar with the details of the MTT test, but some tannins have a high antioxidant activity that may be interfering with the reduction of MTT. Furthermore, if the test is being done on biological material, tannins also has high affinity for proteins and enzymes.Following
- Maddali V S Murali Krishna added an answer:Can anyone suggest a protocol for viable staining of plant cell culture using Evans blue and calculate the percentage of viable cells using UV-spec?
I am working on plant cell culture and I need to find out the percentage of viable cells in the cell suspension using UV-spectophotometer, apart from staining and counting cells using heamocytometer. Please suggest a method.
The followig literature may be useful for your research
The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD(Apoptotic-Like), as it appears that chloroplasts can play a significant role in AL-PCD regulation
Chloroplast and reactive oxygen species involvement in apoptotic-like programmed cell death in Arabidopsis suspension cultures; by Siamsa M. Doyle, Mark Diamond and Paul F. McCabe*
Journal of Exoerimental Botany; 61(2), October 12, 2009,473-482Following
- Shamprasad B R added an answer:How to I dissolve the Bhasma (nanoparticle- metalic powder) if I want to do a cell viability assays in flow cytometry with the hemocytometer?
I have tried to dissolve Tamra Bhasma in water and in DMSO but could not achieve it to 100%. So can anybody suggest a better way to do this?
Thank you Sandeep and Jans Fent for the nice suggestion.Following
- Alisan Kayabolen added an answer:Why I couldn't measure viability of encapsulated cells quantitatively by Presto Blue and MTT assays?
I am trying to encapsulate ASC cells in a hydrogel. To measure viability, I've tried both Presto Blue and MTT assays. However, I couldn't see a difference between values for empty discs and cell-encapsulated discs (5 mm diameter, 2-2.5 mm height). There is a high difference between monolayer controls, by the way, so assays are working. I don't think that this is because cells cannot live in hydrogel, because I am encapsulating cells as 1 million cells/ml and I tried to measure even on the day of encapsulation. Can all cells die that much fast? Also, the pH of hydrogel is 7.4, and I know cells are inside gels since I can see cells as small spheres under light microscope after cutting discs into small slides.
Is it possible that dyes cannot reach inside the gel? I saw some articles in which those assays were used for viability measurements of encapsulations. So, do you have any advices for me?
Many thanks for your answers. I will try your advices if I can have those kits. I hope my all cells didn't die such fast.Following
- Martyn Alun Sharpe added an answer:What is the correct protocol for a live-dead assay in a fluorescence-based well-plate reader using Calcein AM and Propidium Iodide?Can somebody point me towards literature on how exactly a well-plate reader works, and how to perform live-dead assay using staining agents? I am using a 96 multiple well-plate for cell incubation.
I have never managed to get a live/dead method to work with plate reader; although the direct measurement of ATP using luciferase works well in the hands of some of my friends.
The basic problem is that some dead cells give a higher level of staining than living ones and some, non-adherent cells, give less. On top of this we have an unknown level of background staining.Following
- Alarbi Emmakah added an answer:Is anyone familiar with cell (MC3T3-E1) lysis from a PEGDA gel?
I'm doing a cell count, in a 3D structure, to monitor cell proliferation at different time points: 1, 3, 7, 14, & 21 days. I'm looking for the best method to break down a PEGDA gel to be able to access to cell nucleus to preform a DNA assay. However, breaking down the gel network seems to be a challenge. I've done several methods, freezing at -80 c˚/dry, centrifuge, Ultrasonic cleaner bath and eventually I'm trying to use Tris-EDTA buffer solution 100×. I'm wondering, what is the best method to break down a 3D gel network since the PEGDA may not be a reversible gel?
thank you Atanu, that's very helpful!Following
- Niranjan Koirala added an answer:Can anyone tell me the easy methodology to label bioactive flavonoids for the elucidation of anti-cancer activity signalling pathway?
Hello Everyone. I have been working in multiple modifications of flavonoids mainly glycosylation and methylation. We have recently set up an anti-cancer activity testing lab and we carry out MTT assays for several cancer cell lines. Recently we are interested to label active flavonoids and elucidate their binding model and signalling pathway inside the cancer cells. Anyone having experience in labeling technique please do let me know. Thank you.
Hello Xiaoge. Thank you so much for your valuable answer. Could you kindly send me a protocol to track the signalling pathway how and where an anti cancer drug acts?
- Emilia Joanna Orzechowska added an answer:When doing MTT assay in LNCaP, after incubation with MTT for 4 hr, why do some cells get detached while taking out the medium?
I am using Poly-L-Lysine coated 96 well plates. The cells are attached throughout the experiment. At the final step (after adding mtt), some of the formazan were coming out along with the medium while pipetting out the medium....Kindly give me suggestions..
LNCaP cells are not an easy material unfortunately. I had similar observation while I was doing MTT assay on LNCaP cells. I also tried some coated plates or attachment factors but they didn't make thing better. I achieve the best results when I use paper towel instead of aspirating or pipetting the medium out of the wells. Maybe you can try do this in this way. After incubation with drugs you can remove the medium by inverting the plate on ethanol-soaked paper towels instead of aspirating (but very slowly and gently) and do the same after incubation with mtt mixture.
On the other hand there are also viability tests in which you can add a substrate directly to the cell culture medium and after incubation just measure the absorbance without removing growing medium at all.
- Safoura Tatari added an answer:What is the "best" assay for measuring cell viability in chlorella vulgaris cells?
What is the "best" assay for measuring cell viability in chlorella vulgaris cells?
- Emilia Joanna Orzechowska added an answer:Can anyone help with MTT troubleshooting: removing formazan crystals?
I have been consistently running into issues of accidentally removing the formazan crystals following 4 hour incubation of a 96-well plate. This includes adherent and non-adherent cells lines. I have used both a Pasteur pipette under vacuum and pipetting techniques to remove the media and add DMSO. Anyone have any pearls of wisdom to help me refine my technique. It would be most appreciated. Thank you!
In complicated situation I usually turn the plate a little bit (something about 30-40 degree) and use Pasteur pipette to aspirate the solution very gently. Quite important thing is to move it along the well (you can touch the plastic during doing it). On the other hand when I work with the cells wihich not adherent strongly I invert the plate on ethanol-soaked paper towels instead of aspirating (but also gently) like Meghan Ann Rego said. Good luck!Following
- Jiali (Maggie) Zhai added an answer:Does phospholipid interefere with Alamar Blue assay which gives you false positive results?
I have been working with liposomes loaded with cytotoxic drugs. I used homemade resazurin solution (Alamar Blue) to investigate cell cytotoxicity of my drug-loaded liposomes. I consistently obtained false positive results. For example, from Alamar Blue fluorescence reading, liposome-treated cell samples have 200% cell viability when normalized to untreated cell controls.
Thank you for your response, Dzmitry. I did all the controls. To clarify the problem, I compared the toxicity empty liposomes (100nm, composition is DSPC, cholesterol and DSPE) with ACHN cells. they do not contain drugs. I incubated with cells with various lipid concentrations (0-200 ug lipid/ml) and did Alamar Blue assay. Apparently, the liposome generated false positive results (resazurin turned from blue to red), giving me cell viability from 100% upto 300% with increasing lipid concentration. Under the microscope, cells are alive and normal. If there is no interference, shouldn't the cell viability maintain around 100% compared to untreated cell controls?
by the way, I am using home-made Alamar Blue by dissolving some resazurin in PBS. I did untreated cell controls and cell standards to work out the seeding density.Following
- Michelle Rudd added an answer:Does anyone have information on increased cell viability AND levels of caspase?
I've applied a treatment to my cells, and grown them for 5 days. On day 4 and 5 I tested them for cell viability and capase levels. Levels have increased for both of them, this has happened in my replicates of experiments done on different days as well. Does anyone have any idea why? My understanding was that this should be an inverse relationship.
Thanks Bram, that page is very interesting. I have previously used PrestoBlue for my cell viability assay, which works on the Resorufin/Resazurin based principle. That had indicated that my treatment was reducing the viability of my cells, but then when moving to the promega assay I got opposing results. It makes it very difficult. I'm using attached cells. I think I might take your suggestion and try the trypan blue. Thank you, you have been very helpful!Following
- Hardik Suryakant Shah added an answer:What concentration of methylene blue solution should I use to measure cell viability in yeast S. cerevisiae in a toxicity study?
I've been studying toxicity in yeast for a while. I am trying to measure the cell viability. I've tried 0,01 % of methylene blue solution which did not stain any of cell. Then I've tried 0,1% methylene solution in the same sample. At this time, most of the cells (almost 80%) were stained dark blue that means cell are dead. In the literature, I did not find any other concentration and I guess both concentrations gave wrong result for my sample. I am confused about the concentration of the dye that I should use. Is there any recommendation about this subject?
dear, find this protocol
Principle of Procedure used:
The dead cells have increased porosity of the membrane, which allows easy penetration of dyes, and thus they get intensely stained. On the other hand, live cells resist diffusion of the dye because of controlled porosity, integrity and permeability of the plasma membrane. This principle is used here to differentiate live cells from dead cells with the help of dye like Methylene blue or neutral red at a concentration that is non-toxic to the cells. Here, dead cells appear stained blue in color, whereas, live cells appear faintly stained or colorless.
Demonstration of viable cells.
yeast culture suspension
0.5 % Methylene blue solution
Sterile pipettes and test tubes
1. In a sterile tube, transfer 5.0 ml of yeast suspension aseptically.
2. To this, add one drop of 0.5 % Methylene blue solution under aseptic condition.
3. Set the tube aside for five minutes at room temperature.
4. Transfer 3 to 4 loopful of stained suspension from tube on a glass slide.
5. Place coverslip and observe under high power.
6. Record your results.Following
- Vehid Salih added an answer:What is the "best" assay for measuring cell viability in 3D cell culture models?What cell viability assays have been validated for use in 3D cell culture models (eg. spheroids, tissue scaffolds, cells in hydrogels, etc.) and of these, which is the fastest, most reliable, robust, sensitive, accurate, etc.?
For qualitative purposes, may I suggest a simple fluorescent live'dead cell stain such as Calcein AM. I agree with Alamar Blue for a reasonable and reproducible approach for quantitative data.Following
- Pouya Hassandarvish added an answer:Can anyone give me advice on the MTT assay with regards to with or without a medium?I'm using Sigma reagent for MTT test. In our protocol we add MTT, incubate for 3h and solubilise with acidified isopropanol, by adding it 1:1 to the wells, and mixing properly, without removing the medium.
I read there are other protocols where you remove all the medium after incubation with MTT, and use DMSO or SDS.
I am interested to know the reasons for bothering with removing the medium but more specifically the pros and cons of using medium.
some people are removing the medium becoz they want the cell lysis faster and the blue color come out faster and easily can read the absorbance. but other researchers believe removing the medium is disturbing the cells and some of the cells migh go out from the well and that cause error in your assay. Anyway I suggest you to your MTS, is more convinence and you dont need to use Isopropanol or DMSO to stop the reaction. once you add MTS after 1 hor to 4 hour you can detect your plate using ELISA reader.Following
- Anna Jirkovska added an answer:Which is better: MTT or cell viability assay?I would like to determine the concentration of an anti-cancer drug on a cancer cell line to determine the concentration that I have to use to kill only half of the cells. I used a cell viability test before and checked with different concentrations of drugs. Then I choose the concentration that gave a viability around 60%.Hi Eric,
In my opinion, when studying anticancer effect of drugs, what basically you are looking at is antiproliferative effect rather than actual cell-killing, so the difference in the MTT assay results (thus the measurement of cells' metabolism) and the actual cell number obtained by the trypan blue exclusion assay might be negligible in most cases. Although with MTT you should be sure to get proper signal to noise ratio and also make sure the substances you use are not redox active (eg. hydrogen peroxide), as they can interfere with MTT, causing false positives or negatives. Trypan blue is favorable, but rather labor intensive. What I would suggest is to try both methods with some established insult in your cells before, and if you get comparable results, you could go for convenience, or other way around, check your final results with trypan blue.
- Johan Van Meerloo added an answer:Does anyone use the reading of 495nm to assess cellular viability in Huh7.5 cells (MTT method)?MTT methodIts the wavelength where the colour is giving the best od values.Following
- Deepak Kanojia added an answer:Is direct cell counting a viable way to assess cell viability in co-culture after treatment?I subjected a co-culture of cells A and B to a treatment designed to preferentially kill cell type A over cell type B. I used ImageJ to analyze the pictures taken with fluorescence microscopy and saw that there were significantly fewer A cells than B cells when treated with the targeted treatment. Comparatively, there was no significant difference in counts of cells A and B when the co-culture was treated with a negative control. Is this method of counting the cells adhered to the plate a viable way of assessing the effectiveness of my treatment?I perform co-culture experiments using Neural stem cells (NSC) and cancer cells. We simply label the cancer cells with Luciferase. Later we add NSC and at required time point/s simply conduct luciferase activity (which will tell us if cancer cells die). In your case you can label the cells seperately (A and B) with luciferase and conduct a combination of Luc+ (A) and Luc- (B) cells and the other way round, to see which population dies worse.Following
- Marylou Gibson added an answer:What are the best in vivo cell viability assays for virus work?I would like to monitor cell viability during an experiment (in a quantitative way if possible) using the very same cells I'm using for the experiment. I'd be using poly IC and viruses, and would like to make sure that the potential effects are not due to cells dying, but they are actual responses by living -but stressed- cells. One option is to use a Caspase assay on a parallel set of cells, but the ideal would be to use the same cells. What are the best, most used methods to test this?Make replicate wells of your cells and sacrifice a few wells at every time point you wish to test for viability.Following
- Inbal Doron asked a question:What concentrations of the Resistin like alpha should I take for an MTT assay?Has anyone made an MTT assay with the Resistin like alpha gene?Following
- Arun Kumar added an answer:What is the mechanism responsible for the adsorption of Erythrosin by dead cells (bacteria/algae)?Erythrosin is often used in viability assays. Dead cells adsorb Erythrosin and appear pink under the microscope, while living cells are not stained. Can Erythrosin penetrate dead cells because of damaged membranes? On what part of the cell does Erythrosin adsorb?Following
- Stefano Focaroli asked a question:Does anyone have a validated protocol for assessing cell viability on alginate disks?I need a protocol for counting living cells dispersed on a calcium alginate disk that does not involve fluorescent dyes.Following