- Catarina A. Marques added an answer:Can somebody recommend a protocol for Alamar Blue assay using HepG2 cells?
In our laboratory we are trying to determine the toxicity of some compounds against mammal cells, in this case HepG2 cells. So, we have some problems with the Alamar Blue assay, and the culture of HepG2. First, we are growing HepG2 cells with DMEM medium supplemented with 10% FBS and Glutamine. The alamar blue assay was performed with an initially concentration of 10000 cells/well and was incubated with drugs for 48 hours. I’d like to know the time of incubation with resazurin, we have probed 4 and 16 hours, but unfortunately we don’t see any color changes at this time.
Thank you in advance for all the help that you can give me.
Here is a paper that did it in Huh7 cells. At the time the lab was also doing it in Hepa 1-6 (as I did) and HepG2 (I think), and as far as I remember, the protocol was the same.
Hope it helps!
- Annie Bygrave added an answer:How can you maximise the viability of CD4+ T cells when isolating from mice spleen?
I will be getting some knockout mice in a few months and I have some WT mice spleen frozen down. I would like to do a dry run on how to get the most CD4 T cell from the mice spleens. I have very little experience in dissecting mice and collecting spleens so I would leave that to the lab I'm getting them from. Question is, what's the best way to transport them?
Once I receive the spleen, I then need to isolate CD4 T cells. I spoke to some scientific reps and they suggest using the rubber tip end of a syringe plunger and gently push the spleen into a 70um cell strainer, then rinse with buffer.
The instruction seems straightforward but I'm not too sure about the cells viability afterwards. Is there a better way to do this?
Fresh spleen is very soft. Mashing it up with the rubber end of a sterile syringe plunger is the standard, safest way of isolating cells from mouse spleen. The sieve will remove the large pieces of matter and leave you with the cells, which are amazingly undamaged. This is how I personally isolated cells from mouse spleen for several years.
I realise you will be leaving the dissection to the lab you are collecting them from. Even so, if you do need to do it in future, dissecting the spleen is very easy. Having laid the dead mouse on it's side with the left side facing upwards towards you, swab the skin with 70% EtOH. Snip a V shaped flap in the skin of the flank just above the thigh. Grasp the back legs firmly with one hand and the flap of skin firmly with the other and pull smoothly upwards, laying the skin over the mouse's thorax and face. The spleen is a dark red colour and attached to the pancreas (soft, pale, flabby organ) and the kidney by membranes.
You need to gently lift the spleen from underneath without actually gripping it hard with the forceps, because if you grip it, it will break. I used curved forceps with the tips held apart to gently cup the spleen while cutting it free of the attached membranes with iris scissors. Once free, lay it on a piece of clean hand towel and gently roll it over to stick any remaining membrane/pancreas to the tissue before making a final trim to remove this. Rinse the spleen through several changes of sterile medium or PBS.
You could place the spleens into PBS or culture medium and keep them on ice for transport in a polystyrene box with a lid, which you would have to bring back to your lab very quickly. A much better alternative though would be to isolate the mixed population of cells before travel and bring them back in suspension (rather than bringing the whole spleen, which would become hypoxic and degrade much more quickly than cells in suspension).
Frozen spleens are unlikely to yield you complete cells, even if they have been flash frozen in liquid N2. I wouldn't waste your (flash?) frozen spleens on practice. You'd be better off using them for DNA, histology or histochemistry. If you need practice, get someone who has done it before to teach you, using fresh spleens.
Purifying your cells afterwards to isolate the CD4 T cells using magnetic beads or FACS purification is common. This link might be a good start re the magnetic beads process, but I am sure you already have chosen your method. I wish you success.Following
- Roman Mezencev added an answer:What is the different between neutral red and MTT assay?
Is there any condition where I have to use neutral red?
They both determine the same thing, since in both these assays the assay signal is proportional to the number of viable cells (under the conditions of assay linearity). However, both these assays use different approach: neutral red assay (NRA) is based on the accumulation of neutral red dye in the lysosomes of viable cells, while MTT is based on the reduction of yellow tetrazolium MTT reagent by viable cells to purple formazan.
There is a paper reporting that the NRA is more sensitive than several other viability/cytotoxicity assays, including MTT (PMID: 18600217). I would say that NRA can be better than MTT when using cells with inherently low metabolic activity that would produce little formazan. The MTT and similar assays may be also inappropriate if cells are tested for viability upon treatment with inhibitors of certain enzymatic systems. On the other hand, the NRA is a heterogeneous assay and requires washing steps, while MTT, and especially its newer variants (e.g. WST), are homogeneous assays. However, all these assays measure absorbance as an assay signal, which is their limitation nowadays, when highly sensitive fluorescence (e.g. resazurin/AlamarBlue) and luminescence-based homogeneous viability/cytotoxicity assays are available.
Here is a link to an article comparing NRA, MTT and other cytotoxicity/viability assays:
- Andy Wu added an answer:How can I determine cell viability with fixed sections?
I would like to examine the cell viability of my tissue-engineered constructs in regards to distribution. TUNEL staining is an option, but the kits are rather expensive.
I've heard that it may be possible to do typical live/dead stain (i.e. calcien-AM, PI stains) prior to the sectioning. Have anyone tried this before and could you share your experiences with this method? My concern is the preparation step (i.e. washing, fixation, OCT infiltration timing, etc) after the staining and how these factors would affect the final fluorescence signal.
Thanks in advance!
Thanks Gary. I think I will give those a try.
Btw, I typically use 10% neutral buffered formalin for fixation. Would that cause holes in the membrane?Following
- Yasser M. Abd-elrhman added an answer:What are the suitable Cell Lines used for orthopedic implants studies?
I am working on the titanium alloys as biomaterial (Implants), my implants will be in contact with the bone directly.
When I begia to cooperate with one of my biology colleagues to perform invitro study, he asked me about the type of the cell line.
So any one could guide me?
Thank you again Dr SandraFollowing
- Milad Esmaeilbeigi added an answer:What is a suitable way to obtain large numbers of cells for a comet assay?
I would like to prepare a cell suspension from liver and gill tissue.
after cutting tissues(completely) by scissors, Solution produced in microtube was released for ten minute, then 10 microliters from above cell suspension is removed and mixed by LMPA and loaded on slides.
Do you agree with procedure?Following
- Mariangela Garofalo added an answer:How much can I save if don't use the MTS kit and prepare it manually?
Previously I used MTT assay. Now I plan to switch to MTS assay which is more convenient. Does anyone know the difference of pricing for MTS kit if compared to preparing it manually by ourselves? I have quite a number of samples to run with.
Usually I use MTS assay kit from Promega, it's a little bit expensive but really simple to use and also accurate. I have never prepared MTS manually, in the past I prepared MTT solution homemade but then I switched to kit because is easy and also the prize/ quality ratio was better in the kit compared to the homemade solution. Maybe is the same also for MTT.Following
- Deepthi S Nair 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.
@ Ranjith Pathirana Thank you sir for your kind reply. It was really very helpful.Following
- Stanislav Obruca added an answer:Has anyone experience with fixable viability staining of bacterial cells?
I have been searching for some commercially available stains and staining protocols for fixable viability staining of bacteria cells. The viability test would be performed by flow cytometry. Thank in advance for your help.
thanx to all of you for your help.Following
- Jonathan Betts added an answer:Is PrestoBlue® Cell Viability Reagent suitable for MIC determination for Campylobacter?
Is anyone familiar with the use of PrestoBlue® Cell Viability Reagent for mimum inhibitory concentrationson determination in the case of Campylobacter (bacteria)? Is it suitable?
Alamarblue works fine, so prestoblue should too, as the active ingredient in both reagents is resazurin.Following
- Nikola M Stojanovic added an answer:Can you suggest me the protocol for XTT assay?
I have XTT powered substance (not kit!) so I have problem finding a proper protocol for applying this in macrophage/lymphocyte cell cultures.
I have looked at many papers and kit protocols where there are a lot of info about assay but few things are still vague to me! I can't find proper concentration of starting XTT solution nor the exact amount of uL applied in well. The absorbance measured after is done at range from 400-500 nm so which one is the best?
Also do I need the activator for XTT that is stated in all kit protocols but there is no mention about the activator in papers.
Thank you it's quite detail protocol!Following
- Laleh Rafiei added an answer:How can I stimulate endothelial cells by LPS?
I want to stimulate a Human endothelial cell line (HUVEC) by LPS and test my drugs of interest to quantify the anti-inflammatory effect, I used the DMEM medium and 10% FBS for HUVECs, at first I test the viability of cells by MTT assay, for this I stimulated with 1µg/ml, 5 µg/ml and 10 µg/ml of LPS. I just use the 5 µg/ml and 10 µg/ml concentration to see if LPS has some effect on endothelial cells but there was no difference with the control group. Where is the problem? I bought the LPS form Sigma. Does the HUVEC stimulation need a special protocol?
Very thanks Thomas, I will try itFollowing
- Amy Birch added an answer:With regards to, non-specific vascular DAB staining in mice injected with Evans Blue, does EBD bind secondary antibodies?
We have conducted an experiment where mice were injected with Evans Blue (i.v.) and then perfused 1 h later. Brains were extracted, hemisected, and one half was post-fixed (4% PFA, 48 h) for sectioning.
As Evans Blue autofluoresces red, we decided to perform DAB staining to look at various markers (GFAP, CP13, etc..). However, we find non-specific vascular staining with the DAB, even in our no primary controls.
I am assuming that this is the EVB reacting somehow with our protocol - has anyone else seen this? Does the EBD bind the secondary antibodies?
The protocol we use is as follows (on 40um free-floating sections):
10 mins TBS wash, 20 mins hydrogen peroxide (0.6%) quench with Tx-100 (0.1%), TBS washes, 1 h block in 10% FBS in TBS-Tx 0.1%, Primary incubation in 2% FBS in TBS-Tx 0.02% (o/n), TBS washes, Secondary incubation in 2% FBS in TBS-Tx 0.02% (2 h), TBS washes, ABC incubation (45 mins), PBS washes, DAB reaction, mounting & dehydration.
Any comments would be greatly appreciated!
Thanks for all your answers! Let the optimizing commence... :)Following
- Shahla Shahsavandi added an answer:What is best solvent of MTT powder for cell viability assay?I want to assay cell viability with MTT powder from Merck company. What is best solvent of MTT powder for cell viability assay?
I dissolve in PBS at 0.5mg/ml final concentration and filter MTT solution using 0.22um filter.Following
- Elena Yu. Filinova added an answer:Can you answer a few questions concerning the Cell Counting Kit-8 and the MTT method?
- What type of cells can be measured using the Cell Counting Kit-8?
- Is pre-incubation necessary when using the Kit-8? and if so,
- How much pre-incubation time is required prior to the assay?
- What number of cells is appropriate?
- What is the toxicity of the Cell Counting Kit-8 compared to the MTT method?
- Do I have to add trichloroacetic acid in MTT method for a cancer cell assay ?
- How can I determine MCF-7 and Hep-G2 with MTT?
If dissolved in RPMI one would need to make new MTT reagent every time for sure. Cytotocicity test is not being made in 1ml volume (48- or 24-well plates) because reader does not accept these format; to replace DMSO, isopropanol or any other solvent to cuvettes or 96-plate is additional unnecessary step.
Acidified alchohole (isopropanol) is not dissolving cell membranes completely, it is fixator. So, absorbance rate will be partially lost in formazan which would not leave cells, and another portion of absorbance color - in not removed medium. The same happens with trichloracetic acid usage I think.Following
- Reza Heidari added an answer:What should be the percentage of viable cells in a particular tissue to carry out the Comet assay?
In a tissue sample, I got only 70% viable cells. Is it acceptable to carry out Comet assay in this tissue sample?Following
- Marzieh Mahdavipour added an answer:Is Low melting point agarose and agarose having low EEO the same?
Can I use agarose having a low EEO instead of LMPA in comet assay? Will it give positive results?
I did this experiment once. But it stopped at the first step!! Because it jellified faster than LMPA and I couldn’t make the cell suspension ;-) if you pass this step, I would appreciate you to share your experience.
- Amit Kumar Mishra added an answer:How is a grooving curve made for determining cell seeding density?
I am new to cell culture. I have been trying to standardize my MTT assay for two cell lines, but the seeding density is not clear and this affects reproducibility. Please advise.
Seed with the density of 10,000 cells by counting with the hemocytometer in 96 well plate and add the compound next day and incubate for the planned period (24,48,72,96hrs). 80-90% confluency is good.Following
- Divakar Selva 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..
Thank you for the suggestion, I have followed your suggestion and optimized the procedure.Following
- Mahbobeh zamani babgohari added an answer:Does anyone have a protocol for cell viability assay in which no absorbance measuring is needed?
I need to measure cell viability in 48/24 well plates. However, since the cells are under treatment of an organic material which has some absorbance itself, I can't use/ trust the optical density of the culture. I would be appreciated if you suggest a method (not trypan blue) by which I can measure the cell viability in well plates.
thank you all for your helpful ideas. that's great.Following
- Mohammed Alhaj added an answer:Among DMSO and MTT dissolving solution (containing HCl, isopropanol and SDS), which is better in an MTT assay?
Some studies have shown use of DMSO to dissolve the formazon crystals, while some have used MTT dissolving solution. DMSO is rapid and produces colour within seconds, while using MTT dissolving solution may need some time for getting proper OD readings 0.75-1.25. Which is reliable? Please suggest.
both DMSO and isopropanol/HCl gave a color immediately after added to cell culture
but before read the absorbance it is better to shake plate for 5 minutesFollowing
- Eric Cox added an answer:How can an MTT assay be used for growth potential assessment?
The MTT assay is usually done for studying the viability of cells by treating the cells with a particular drug in serial dilutions.
With this limitation that if the product you add also influences metabolic activity. The outcome of the test won't be a measure of growth but of metabolic activity and proliferation.Following
- Mohammed Tanjimur Rahman 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!
I used to have same problem....then started hypodermic syringe to remove the Media+MTT which worked perfect.Following
- Amar Bahadur Singh added an answer:Serum starvation in cell line treatment?
How long after seeding of cell line, should I do serum starvation or serum 4%? 24 h or less? How long should it last (before treatment) or serum 4%? 24 h or less? i i want to do serum starvation or serum 4% after and before treatment of B65.
generally we do overnight 0.5% serum.Following
- David J Walker added an answer:Does anyone know if it is possible to peform a dual-stain of cFos and TUNEL?
I am planning to perform organotypic slice culture experiments and I was initially planning to perform a TUNEL assay to test if the neurons in my cultures will survive. However, I was wondering if it was possible to carry out immunohistochemistry on the slice by incubating in an antibody against cFos at the same time. Does anyone know if is this is possible? Thanks in advance.
Thanks Goodwin. I found a TUNEL TMR red kit that has been validated in my animal model so might go with that.Following
- Arvind Singh Chandel added an answer:Why, in MTT assay, do we measure the optical density (OD) at two different wavelengths?I've been reading through some protocols and also come across other journals which only take a reading at a 570nm wavelength. Is it wrong (or is the data valid) if someone hasn't read the absorbance at 630nm? Could anyone please advise me the conditions under which I should read the reference wavelength of 630nm?
Thanks for suggestionFollowing
- Anish Nag added an answer:Is it possible to have both a reduction in cell viability and cytoxicity?
I thought this should be an inverse relationship but I have seen this decrease in viability and cytotoxicity when comapring my treatment samples to my untreated control, on three seperate occasions. I am treating cells with folate.
oxidative burst can increase the metabolic activity of a cell resulting in high assay value (e.g. MTT) i.e. apparently low cytotoxicity actually highly cytotoxic and leading to obvious cell death.Following
- Lidia Mazur added an answer:Are formazan crystals from MTT assay stable for long period?
I wonder whether I can aspirate the MTT solution and let formazan crystal dry overnight before adding DMSO. Would this influence the reading and result?
1-3 hr after dissolving the formazan crystals with acidified isopropanol (0.5 NNaCl in absolute isopropanol), absorbance of the obtained solution was usually measured using spectrophotometer (a at a wavelenght of 570 nm)Following
- Monika Werdiningsih 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.
Thankyou for ur answer, but do you have or know any textbook or journal that said so? I really need support references. I'd love if u have or know it :)Following
- Bashir Alsiddig Yousef added an answer:What is the best caspase-3 Colorimetric Assay Kit?
what is the best caspase-3 Colorimetric Assay Kit?
we are using Caspase-3 Assay kit (Beyotime Institute of Biotechnology, Nanjing, China) and it provide to us good results. the concept of the test is depend on production of spectrophotometric detection of the chromophore p-nitroaniline (p-NA) after cleavage from the labeled substrate DEVD-pNA.Following