- Shanshan Wu Howland added an answer:1Problems with Annexin V-PE / 7AAD compensation on cell viability assays measured by flow cytometry?
I am trying to do a cell viability assay with Annexin V-PE and 7AAD, analysing by flow cytometry.
I had 2 single color controls with a cell population that I knew would be partially positive (one control stained only with anti Annexin V, another one only with 7AAD). After compensating with FlowJo using a strategy similar to that (https://www.youtube.com/watch?v=V4hlIHBJRvE), I saw that most of my values for 7AAD were near zero.
In the compensation Matrix, I saw that I had a spillover of around 140% of PE on 7AAD channel. So maybe after the compensation I lost all my signal for 7AAD.
I searched for answers and a lot of people suggest to use other fluorophore combinations, but for what I know Annexin PE/7AAD is quite a "popular" choice, isn't it? I don´t know what I could do.
Gating details: My first gate selected the cells of interest excluding the debris on the SSC x FSC graph. I also saw other people saying that it would be nice to do a negative selection gate on the debris. Is it just another way of doing it, or the negative gate is required? I'm new in flow cytometry/compensation issues, so any help would be appreciated. Sorry if the question is nosense or too naive.
Cells that are dead will typically have higher SSC and slightly lower FSC than live ones. So your dead cells may now be in what you think is the debris. Take a look at Fig 4 in the link below.
The second issue is with regards to your single color controls. If you used healthy cells to make those controls, very few cells would be positive for either Annexin or 7AAD, and your compensation wouldn't be good. You need to do something to induce apoptosis and/or death.Following
- Cássio Prinholato da Silva added an answer:3Anyone expert in Penicillium chrysogenum, how can I count total cells and measure viability?
Anyone expert in Penicillium chrysogenum, how can I count total cells and measure viability? Thanks
All the best for you!Following
- Mohammad T. Raad added an answer:3Polymer coated Fe nanoparticles are insoluble in various organic solvents. How can I prepare these samples for cell viability assay?
Fe nanoparticles coated with polymer have been developed as drug carriers. Particle size <50 nm.
They are highly insoluble in organic solvents including DMSO or Ethanol.
Due to their solubility issue, their dilution is also a hassle.
We have tried sonicating the samples prior to dosing the cells.
Everytime the result is "nontoxic" even with dose as high as 2mg/ml.
I doubt that the nanoparticles are not interacting with the cells to manifest biological effects.
Before applying the Polymer coated Fe nanoparticles to cell culture, the nanoparticles should be well distributed in DMEM with 10% heated inactivated FBS freshly added using ultrasound treatment in order to obtain nanoparticle colloidal suspension. These nanoparticles exhibited stable chemical and thermal properties.Following
- Riccardo Gottardi added an answer:5How can I conduct Live/Dead Assay in cell seeded hydrogel?
I have some problem here. I used Chitosan Alginate hydrogel in my experiment, and I seed cells onto it.
what is the best procedure to conduct live/dead assay to cell seeded hydrogel? Since the hydrogel itself absorb the staining? Thus, I can't really determine which one is the cells/hydrogel.
If you hyrogel is thick enough, cut it in half using a scalpel so you can see a cross section and the cells inside. Treat the cells with the live/dead assay as indicated by the lifetechnologies protocol but increase the overall concentration of Calcein AM and ethidium homodimer by 4x to 10x compared to what is listed in the protocol. incubation time of 20minutes to 30 minutes is sufficient. Place the flat cross section part of your hydrogel on a glass slide, keep it hydrated and image. NOTE, in 3D you can only see a few cells at a time within the focal place, so most of your cells will be a little "blurry" because they are out of the focal plane. This works well for a number of hydrogel, I have tested it on collagen, gelatin, alginate, native tissues, ECM-derived hydrogels, etc.
An alternative is the use of a glucose assay or and MTS assay to assess cell viability. You will not get the amount of dead cells but only of the live ones, but it is quantitative. I would generally recommend to perform both, a Live/Dead assay and a viability (metabolic) assay on identical constructs.Following
- Gary Lee Gilmore added an answer:4Regarding the MTT assay; shall I process my results or perform a cell viability assay?
I did an MTT assay on HT-22 cell line of my drug. I got the higher reading in my treatment sample, it shows that the drug is proliferating the cells, because the reading becomes higher, when I increase the time of treatment.
I want to ask, how I should show it in the bar diagram, or should I go for the other cell viability assay to confirm?
MTT actually measures metabolic (mitochondrial) activity, not proliferation directly. Perhaps your drug is altering the metabolism of your indicator cells. I agree that a dose response and a time course are good ways to determine if your drug is actually increasing proliferation or inhibiting apoptosis. Checking the viability by trypan blue is definitely worth doing.Following
- Yagya Subedi added an answer:12My MTT reading is fluctuating.. can someone help me?
i have performed MTT as per Mosmann T on HepG2 cell line using different concentrations of plant extracts (1-90 ug/ml). The absorbance of all the treated groups are reduced to about half as compared to the control (untreated) wells. However, the absorbance is fluctuating instead of decreasing with increasing concentration of the extract. what could be the reason? and what can i do to solve this problem???
If your compound is reducing compound then you might need to do the blank test keeping only compound in medium (without cell). This will give you the idea weather you can use MTT assay or not. If your compound is not reducing compound and still result is fluctuating then there might be problem in number of cell in each well plate.Following
- Kriengsak Lirdprapamongkol added an answer:19I ran my MTT assay twice and get no results, can someone please assess my method and help to correct the errors?
1.After 48 hours of adding my compound(100 ul) in 100 ul medium containing 1000 cells ( Human lung small cell carcinoma NCI H82) per well, I then removed all the medium and added 20 ul of MTT solution( 5 mg/ml prepared in PBS).
2. Incubated the plate in water bath at 370 C for 4 hours.
3. Added 200 ul of MTT solubilasation solution and incubated in water bath at 370 C for 30 minutes.
1. The cell number per well is too low, it should be around 4,000-10,000 cells/well for incubating 48 h with test compound.
2. Media containing MTT should be freshly prepared before assay, by mixing 1 vol of stock 10X MTT (5 mg/ml in PBS) with 9 vol of culture media, final is 0.5 mg/ml MTT in media. I use 100 microL/well, if your plate has 60 wells, 7 ml of the media containing MTT should be prepared by mixing 0.7 ml stock 10X MTT + 6.3 ml culture media.
3. At the end of treatment, after you remove the media from wells, immediately adding 100 microL/well of the media containing MTT (0.5 mg/ml) and further incubating the plate in a CO2 incubator (not water bath) for 2-4 h (depend on cell line) to allow formation of purple formazan product. No need to wash the wells with PBS before adding MTT, it will wash out the weak cells away and you will get the over-cytotoxic results of your test compound.
4. To lyse the cells and solubilize the purple formazan product, remove the media out and add 100 microL of DMSO then shaking the plate on shaker around 3 min. Measuring absorbance at 550 nm by using a microplate reader.
Note: some cell line e.g. HT-29 die easily it you left it dry during you are removing media from the wells, so add the media containing MTT into the wells immediately. But most of commonly used cell lines e.g. A549, HepG2, MCF7 can tolerate until you finish removing media from all wells in the plate.
I use this protocol for several years, you can look at in my paper. "Curcumin suppresses vasculogenic mimicry capacity of hepatocellular carcinoma cells through STAT3 and PI3K/AKT inhibition" Anticancer Res. 2014 Apr;34(4):1857-64.Following
- Cássio Prinholato da Silva added an answer:3Any advice on cell test with evaporable aldehydes (formaldehyde, acetaldehyde and acrolein)?
1) Acetaldehyde and acrolein both are very evaporable and there are some loss during the solution preparation and incubation with cell in the 96-well plates or other plates. So A549 cell survival rates ( or other test endpoints ) always show different (or inconsistent or ruleless) results under the same test condition in the different repeated tests. Aldehyde solutions were made with the current use! It is very dangerous to operate with aldehyde solution, especially when adding this solution in the 96-well plates or other plates due to their evaporable nature. Do you have the same experience in the similar experiments? And how do you deal with it? Could you give your some good advice or your good experience to me?
2) Maybe as what we have saw, it is obviously to find the culture medium is seriously metamorphic when using the culture medium (1640 medium is used to feed A549 cell by us) to prepare the stock solution of aldehydes. Can the results be true and accepted under this situation? Or may you have some different methods to avoid this situation?
I wish all the best in your researchFollowing
- Kriengsak Lirdprapamongkol added an answer:9How can I determine cell viability under hypoxic condition?
i want to determine viability of cancer cells under hypoxic condition. i want to know that after trypsinization of cells growing under aerobic condition, can i plate the cells and incubate immediately under hypoxic condition for about 24 to 48 hrs or should allow cells to get attach under aerobic condition and then transfer the plates in hypoxic condition? After the incubation period, when MTT containing media is added, whether the plates should be incubated under aerobic or hypoxic condition?
In the Oncol Rep 2013 paper, the test compound was incubated with cells for 30 min before subject to hypoxia, in order to ensure that the compound can reach its targeted proteins inside the cells before starting hypoxic response.
If your test compound is small molecule, there should be no difference between immediately subject to hypoxia and 30 min incubation before hypoxia.
But if your test compound is large molecule or nanoparticle, it should be incubated for a period to allow the test compound is uptaken by endocytosis and released into cytoplasm before starting hypoxic response.
In another paper, I use a different treatment schedule. See in "Curcumin resistance induced by hypoxia in HepG2 cells is mediated by multidrug-resistance-associated proteins" Anticancer Res. 2012 Dec;32(12):5337-42. The cells were pre-incubated under hypoxia for 24 h, then bring it out for drug treatment and immediately return it to hypoxia for further 24 h incubation. For some compounds, the pre-incubation under hypoxia for 24 h before treatment gives results different from treatment before starting hypoxia. Because pre-hypoxia can up-regulate drug transporter genes that can pump-out the compound. This pre-hypoxia model mimics the situation that patients already have hypoxic tumors before drug treatment.
Good luck ;)Following
- Jeann Murray added an answer:4What is the acceptable range for the raw data readings from the MTS assay?
Just a summary of what I've been doing: I have been conducting the MTS assay on MCF7 and PC3 cells, and my readings from the vis spectrophotometer for PC3 in the absence of an inhibitor (that is, my positive control) is usually 1.0 - 2.0 but for MCF7 it is usually 0.7-0.9. For both cell lines, I add 1.5 X 10^6 cells in 20 mL for each plate that I set up, then incubate for 24 hours, after which the test compound and incubate again for another 24 hours. Then I wash the cells once with PBS, add 100uL of medium and 20uL of MTS solution and incubate for 4 hours. My blank/background (that is, wells without cells or inhibitor) is usually around 0.16-0.2 for both cell lines.
The issue is that the person who trained me (he works abroad, but visited my country for a short time for training) insists that anything below 1.0 is too low. Perhaps, as long as you have a specific concentration of cells in the positive control, the readings should therefore be consistent no matter what the cell type is. But is that really the case? Or is the level of the reading dependent on the cell line under investigation?
Please let me know your personal experiences. Thank you.
Thank you so much. Frances, I did think of doing that before so I am relieved to hear that suggestion and explanation in terms of difference in metabolic activity. My MCF7 cells grow slower than my PC3 cells......PC3 needs subculturing every 3-4 days whereas MCF7 is usually every 5 days. So it made sense to me that the MCF7 readings were lower than those for PC3.Following
- Olivia B Harris added an answer:3Has anyone used the ArC™ Amine Reactive Compensation Bead Kit with fixable viability dyes from other vendors?
I received a sample of the ArC™ Amine Reactive Compensation Bead Kit from Invitrogen, but we currently use the eBioscience/Affymetrix line of fixable viability dyes for live/dead cell discrimination for flow cytometry. For compensation of the viability dye, we normally use heat treated cells which works fine. It would be a huge time saver if we could use a bead kit for compensation but I don't want to switch over to the Invitrogen line of viability dyes until we have used up our current stock. So has anyone ever used this bead kit with the eBioscience dyes?
I would agree with Allison - since they are both amine reactive then I am sure it would work if you need to use up your current dye. I have previously used the invitrogen live/dead with the comp beads and can vouch for their utility! Good luck.Following
- Parijat Sarkar added an answer:12How do I normalize my MTT assay for reduced cell number in treated sample if my drug inhibits cell division?
I am doing a MTT assay where because of drug treatment , the no. of cells in treatment is reducing to half of control. Now, after MTT assay how do i know the reduction in absorbance is because of reduction in no. of cell or reduction in viable cells or combination of both?
I feel there are two ways to deal with it:
(1) I plate double the no. of cells in treatment well, so that after treatment the cell no. is comparable with control?
(2) I do a calibration plot with different no. control cells and then compare my drug treatment case.
Which one is more appropriate?
Thank you all for your valuable suggestions. I will do the experiments and post the results soon !Following
- Baltramiejus Jakštys added an answer:7In MTT assay a cancer cell line growth activity (untreated control cells) is decreased in repeated measurements. Why?
We usually use MTT assay with more cervix carcinoma cell lines to detect antiproliferative effect of drugs. At the end of these measurements we get absorbance values according to produced formazan crystals what is proportional to cell viability. The measured values of treated cells are always compared to values of untreated control cells. In general, the measured absorbance values of untreated controll cells (which are comes from one cell line) are constant. In the case of C33A cancer cervix carcinoma cell line the absorbance values of controll cells are about 2700 (usually between 2500 and 3000, according to formazan producing of spreaded cells in plate, 10.000 cells/well). Nowdays I experienced that these values decreased, only about 2000 in experiments. It refers a strong decreasing of cell growth activity in the case of C33A cell line. What is the reason of this decreasing? Have somebody ever experienced similar growth activity decreasing on C33A cell line? Thank you for answers!
Hi, Andras. First of all, I would like to ask how you get 2000-3000 values on absorbtion when it is assumed that getting more than 1 in absorbance is already an unsignificant value (or you talk about cell number which was estimated by using calibration curve?). So, the values of absorbance should be below 1 firstly. Secondly, why do you dissolve or dilute MTT in DMSO? We dissolve MTT in 1x PBS. Moreover, what is the rate o cell proliferation when unaffected? I ask in order to understand if cells do not overgrow during the period you choosed.
Going further, read my article, you will understand, that first of all, you measure cell methabolic activity of cells since MTT is based on methabolic pathways, rather than cell number. Scientists trying to get easier results started to use method for cell viability to be measured indirectly, but if you affect cells by side effect, you will decrease or increase cell methabolic activity according to the type of the side effect which will lead to misinterpretation of cell viability.
In order to answer your question i would comment this way: i never get the same values of different repetitions of the same experiments. What we do, we always take control and normalise the results to control. The results of absorbtion tend to jump higher or lower in my opinion due to different cell methabolic activity that are used for experiment. This may definitely be affected by a numerious attributes- cell size, stage of cell cycle, trypsinisation etc.
- Niels Haan added an answer:6Has anyone tried altering the suggested dilutions given in invitrogen Click-iT EdU Imaging Kits?
I have read that (as usual with kits!) the amount used can be significantly dialled down to make the kits go a little further. Does anyone have experience with this?
No idea what the concentration is, but I know it's more concentrated in the final solution than 1 uM, as you get a greenish tinge to the solution at the recommended concentration, but not at 1 uM.Following
- Ali A R Aldallal added an answer:4How can I dissolve a drug in DMF?
I purchased a drug (CX5461) and the data sheet said to dissolve in DMF. I purchased 5mg, calculations showed I needed to add 486ul to my 5mg powder to make a 20mM stock. When I added the DMF the powder would not dissolve and was still clumpy after vortexing too.
I then decided to dilute my stock further with DMF to a 5mM stock, after vortexing and sonication it looked as though it had finally dissolved.
When I went to take 160ul from my stock to prepare my first concentration of 100uM (in fresh tissue culture media) the dmf+drug mixture just turned into a white milky plasticy solid when I tried to depress from the pippette...
I am not sure if the DMF reacted with some of the plastics I was using or If i did something wrong somewhere, If anyone could please shed any light on this problem I would greatly appreciate it,
Try to use this link may be helpful.
- Ingvild Rognmo added an answer:4How may I analyze drug sensitivity in differentiated neuroblastoma cell lines?
I have the following issue:
I am currently working on the role of a miRNA (I will call this miRNA miR-X) on drug sensitivity in neuroblastoma (see figure pdf).
I found that this miRNA significantly induces differentiation in the neuroblastoma cell line that I am using.
In order to analyze whether the miRNA increases sensitivity to anticancer drugs, I had planned to use the AlamarBlue assay to measure cell viability. The alamarBlue assay is based on that living cells are metabolically active and are able to reduce the non-fluorescent dye resazurin to the strongly-fluorescent dye resorufin.
However, now I am speculating whether the alamarBlue assay is the “right” assay to measure sensitivity of cells to a certain drug in my case.
I found that transfection of cells with miR-X mimics significantly reduces cell viability (50% cell viability in miR-X transfected cells 72 hours after transfection- see figure pdf).
But I am now raising the question whether miR-X – transfected cells cells are really less viable as compared to control-transfected cells?
Does the alamarBlue assay rather indicate decreased proliferation and metabolic activity due to differentiation?
Can I really use the alamarBlue assay to evaluate whether cells are more sensitive to anticancer drugs?
Which assays may I use? I will analyze apoptosis by Flow cytometry and/ or western blot analysis. Do you have other ideas?
Some of the drugs that I am using induce mitotic catastrophe and not apoptosis. How may I measure drug sensitivity in this case?
My former research group measured mitotic catastrophe in neuroblastoma cell lines and also in patient biopsies that we had injected into mice.
We embedded the tissues in paraffine, made thin sections, stained with DAPi and analyzed the sections in the microscope where it is easy to identify mitotic catastrophy just by looking at the cell morphology. Then you can just count dying cells in treated samples and in control samples to get a ratio.
We have just published a paper if it is of any interest to you.
Let me know if you need more info.
- Carlos Sonnenschein added an answer:10Can MTT assay be taken as equivalent to cell proliferation assay (BrdU)?
Im currently working on SH-SY5Y cell line. The treated group showed increased BrdU +ve cells on Immunocytochemistry technique but % cell viability from MTT assay showed no significant effect. If there's increase in cell proliferation, % cell viability should also increase, at the same time period of treatment. I would really appreciate your help analyzing this case. Thank you.
Dr Bennett's is the comprehensive answer. Use instead a particle cell counter (Couldter Counter or alike).Following
- Amanda Villalvilla added an answer:8Does the optical density of formazan dissolved in DMSO change over 24 hours?For ease I would like to perform an MTT assay and dissolve the formazan in some of the wells of my plate while other wells continue to be treated at 37 degrees overnight. By the time I have added MTT to the final treatment condition and I solubilize the formazan, it will be close to 24 hours since I solubilized the first wells. Over the 24 hours, is it likely that the optical density will change significantly enough to prevent a fair comparison of the two conditions?Following
- Hugh J Byrne added an answer:2Which cell line is best for Solid Lipid Nanoparticles Skin cytotoxicity?
I am plannnig to perform skin toxicity studies for my solid lipid nanoparticles for skin delivery. I am in a fix which cell line i should use? L929 mouse fibroblast cell or mouse 3T3 fibrobalsts cell...?
Similar to Claudia, we also use HaCaT cells. Skin models are expensive, and be careful about their use for permeation studies.
“Comparison of Structure and organization of cutaneous lipids in a reconstructed skin model and human skin: spectroscopic imaging and chromatographic profiling”,
Ali Tfayli, Franck Bonnier, Zeineb Farhane, Danielle Libong, Hugh J. Byrne, Arlette Baillet-Guffroy,
Experimental Dermatology, 23, 441-443 (2014)Following
- Jakub Wojcieszak added an answer:6Why do MTT and XTT assays give inconsistent results?
I have a problem in my cytotoxicity testing.
I tested one compound on 3 various cell lines with MTT assay and it gives concentration-dependent decrease of mitochondrial function (by around 60% at the highest concentration) on all 3 cell lines.
However, when confirming my results with XTT I see no effect, or even a slight increase of substrate reduction (by around 10%).
LDH assay also points no increase in the number of dead cells.
I repeated MTT with this compound in modifications of the method:
- Adding MTT into the medium -> incubation -> aspiration -> solubilization in DMSO -> shaking -> reading
- Aspirating the medium -> adding MTT -> dissolving in DMSO -> shaking -> reading
... and it always gives the same effect, so I guess it is not chemical interaction between my compound and MTT itself.
Does anybody have an idea what can be the cause of this inconsistency? I know that MTT utilizes NADH to reduce, while XTT uses NADPH, so is it possible that my compound selectively lowers NADH concentration or inhibits enzyme responsible for MTT reduction?
I extended the incubation time to 72 hours and it resulted in stronger decrease in MTT and strong increase in XTT (153% of control group). Strange ...
For now I am planning to measure intracellular total and reduced glutathione levels and mitochondrial membrane potential with JC-1.
Do you have other suggestions or possible explanation of what I am experiencing?Following
- Petr Mlejnek added an answer:13Can % viability be considered an accurate representation for proliferation measure by MTT assay?
My MTT assay shows proliferation in treated microglial cells as compared to noemal control. If one mesures using %viability, the values for the treated group come to 150% relative to the normal controls. Can the be considered as a valid representation? If not, what are the alternatives? should a direct representation of absorbance work fine?
Also, could anyone suggest a better and more efficient proliferation assay, that is cost effective too.
No. % viability means to discriminate live and dead cell usually based on permeability of cell membrane. This measurement fails to uncover early apoptotic cells which are in fact dead (but have intact membrane). This is really huge problem! On the other hand, MTT assay is not able to distinguish between the number of cells and the decreased viability. The decreased value of MTT reflects either the decreased number of cells or the cell death, or very often both events. However without further analysis you do not know what has happend.
- Khatereh Khorsandi added an answer:5In my MTT test, when I use higher concentration of drug for treatment, there is higher absorbance than in the control. Can anyone help me?
In my MTT test, when I use higher concentration of drug for treatment, there is higher absorbance than in the control. Can anyone help me?
thanks all, I made negative controls for all experiment but now I think the problem may cause from color of drug.Following
- Hoda Abolhasani added an answer:16Cells treated with DMSO show less viability than cells treated with the compound, how is that possible?
I have performed a MTT viability assay to see how my cells (MCF-7 and HCT-116) respond to a drug. I had one control with cells + medium and another control with cells + medium + DMSO in different concentrations, the cells treated with the compound (wich is dissolved in DMSO) showed more viability than the cells treated with DMSO only in the same concentration. How is that possible? Is that normal to happen in a MTT assay?
- Hussein A Abbas added an answer:4Any advice on the UV to inflict DNA damage to HEK293 cells in culture?
I am studying a protein and from imaging I can see that my protein is recruited to sites of DNA damage. I wish to UV irradiate HEK293 cells in culture prior to collection and analyses by RNA-seq and mass spectrometer. Does anyone have an idea of how to (protocol and instrumentation) UV irradiate cells in culture for such studies?
I think an important issue to consider is whether you are interested in acute or chronic changes. If acute, you want to plan your experiments to maybe collect the cells within 6 hours. If you want to see changes over time, that's a different issue. We have done similar experiments in the lab but with different cell lines-Following
- Coral Sh added an answer:6Can DCFH-DA cause cell death when added to measure ROS level?
I have been trying to measure ROS in mesothelioma cell lines using DCFH-DA. But every time after adding DCFH-DA to the cells even at 10 micromolar, the cells would become rounded and detach after 15min of incubation. I tried to reduce to 1 micromolar and then 0.5 micromolar. The cells were ok but the fluorescence is too low. Has anyone encountered the same problem?
Thank you so much for all the comments and suggestions.Following
- Adeola Folasade Odewusi-Ehigie added an answer:22Which 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%.
I'll go for CCK-8, It's very easy to use, sensitive and straight to the point. With it you can determine the viability of your cells at varied concentration within a limited period of time. However, the Kit is expensive. In the alternative you can use MTT Assay which will take an extra hour before you acquire your results. All the best!Following
- Gregory J Brewer added an answer:3What cell viability assays for low concentration neuronal culture?
I want to determine the level of toxicity of some treatments on neuronal cultures with a very low concentration (from adult animals - plated on Poly-Lysine coated coverslips - between 100,000-200,000 cells/ml max ).
I already tried LDH assay kit but it seems that the density of cells is too low.
I read that Alamar Blue or PrestoBlue are working with low concentration of cells. Anyone has any experience with that in neurones at low density?
Also, as I am not able to plate my neurons in 96 well-plates (because they don't stick to plastic), I need to do the test in 12 well-plates containing coverslips with my neurones. And I am afraid the bigger volume is diluting the LDH (or maybe the Alamar blue as well). Do you have any opinion about that?
For years we have used a single cell fluorescence assay based on propidium iodide stain of dead cells and fluorescein diacetate for live. See our 1993 paper or buy Invitrogen's Live-dead kit for 100x the price.Following
- Falugi Carla added an answer:7Is it possible for a cell to be viable while being PI positive?
Propidium iodide (or PI) is an intercalating agent and a fluorescent molecule with a molecular mass of 668.4 Da that can be used to stain cells. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm. Excitation energy can be supplied with a xenon or mercury-arc lamp or with the 488 line of an argon-ion laser. Propidium iodide is used as a DNA stain for both flow cytometry, to evaluate cell viability or DNA content in cell cycle analysis, and microscopy to visualise the nucleus and other DNA-containing organelles. It can be used to differentiate necrotic, apoptotic and normal cells.
If you need to introduce some molecules inside a living cell, you can try to use liposomes. When PI is inside the cytoplasm, it can stain also mRNAs.Following
- Panduranga Rao added an answer:4How do I monitor bacterial viability in vitro?
I would like to investigate the intracellular viability of pathogenic bacteria within a host mammalian cell. Is there any relatively simple way to do this without lysing the host cell?
CFSE is a good idea if you want to look for replication (cell division). In case of intracellular bacteria probably it is of limited use as half of the divided bacteria may not come out.Following
- Puszko Anna added an answer:4How to understand the concentration of a compound in articles when I read about proliferation and viability assays on cells?
I am a chemist but I need to learn how to make some cell assays. I don't understand one thing; in articles I can found concentrations of tested compounds used for assays in e.g. 5 ug/mL or 5 uM. Does it mean that the concentration of the tested compound was like this in each well or is it a concentration of a solution of this compound used to make the assay, which means that the real concentration in the well is different because I added only a small portion of the prepared solution into the well with cells in the medium?
This question might sound silly, but no one around me knows the answer...
Thank you for any help.
Thank you all for fast reply and good explanation!!! Now I am sure that my thinking was correct. :)Following