Trypan Blue - Science topic

Hi,

I am looking for a viability marker to stain alive white blood cells and still working after fixation.

Here is my workflow for you to better understand (and tell me if my asumptions are wrong).

1) I am studying white blood cells from total blood (EDTA tube).

2) I take a certain volume of blood and had a buffer with fixative agent during 8 min (blood is diluted in 1:1).

3) Then, I make the blood go through a filter to stick fixed cells on it.

4) Finally, I stain the filter with May-Grünwald Giemsa - MGG (resulting in colors from pink to blue with purple) to see the nucleus, cytoplasm et identify my cells under microscope (brightfield).

My idea is to be sure that my cells I identify by MGG were well alive before the 8min fixation.

--> So, I would love to have a viability markers that I could identify in brightfield (so more a dye?), in different colors other than pink/blue/purple and that is not toxic for cells (no apoptosis or death process). If you have an idea.. let me know please :)!

I have made some research and here I found :

Thank you for you returns! :)

Hi all,

I am having trouble deciding if this is cell contamination or something else. I keep seeing this fiber like bodies in my media (not on the same level as my adherent cells). There is no smell or media color change that indicates contamination. I do also see these fibers when i attempt to do a cell count with trypan blue. Has anyone seen something similar? I dont see any movement at all and they dont move when I jiggle the flask gently. I am concerned since I keep seeing these even after I rinse with PBS and place in new media. Is something off with my sterile filter when making my media or is this some type of yeast or bacterial contamination?

Hi all.

I hope to learn which companies' (sigma, thermo scientific, etc.) cell viability kits or reagents are popular for cancer research. Specifically, I would like to know about MTT, CCK-8, LDH, alamarBlue/Resazurin, trypan blue, and sulforhodamine B kits. It'll help a lot for my market research on which kit to use for OSCC cell lines!

Hello!

Have you ever observed that when measuring the viability of freshly thawed PBMC with trypan blue, the value is higher than with Annexin/PI staining or some other staining that takes into account early apoptotic cells? if yes, how large was the difference between the two methods? Which method is more reliable and will tell me the actual survival rate of the cells?

Thank you!

Hello everyone.

I am looking for suggestions for HL60 cell line culture. Our lab is new to suspension cell culture. we bought the cell line from National Center for Cell Sciences (NCCS) Pune. Upon arrival, the cell was absolutely fine, having a doubling time of around 48 hours and we were able to freeze 10 vials with 10^6 cells/ml (90% FBS + 10% DMSO). But after 6 months in liquid Nitrogen when we started culturing from those vials it seems that the cells are not proliferating. We are seeing cell clumps forming and after 3-4 days all of these cells are dying (when checked using trypan blue). Can you help me with this problem? We are unable to point out the exact cause of the situation.

Thanks

I have cultured PBMC and some RBC contaminant for 2 weeks in suspension and low attachment plate. From the very first day, I noticed that there are many cells that have already turned all black-ish or grey-ish color. I don't know why and I keep continuing my culture process because the sample is very limited and I don't want to waste any samples. This is very confusing as because I will do viability assay using trypan blue, but the cells were already black. So I can't see the blue-ish color that supposed to appear. For additional information, my medium is DMEM F12, EGF, FGF, CoCl2, ITS, Penstrep, and Amphotericin.

In the picture derived from light microscopy, there are some red stains apart from the blue ones. I am wondering, what could it be?

Currently I am trying abcam protocol for it.i checked the cell lysis by trypan blue stain which is more than 90 %. but I am facing following issues

1. unable to remove beta actin in mitochondrial fraction.

2. I am able to see mitochondria pellet (cell-1-1.5 X 10^7 cells) but the protein yield is very low 30 ug. and band of interest-SDHA is also very less on western blot compared to whole cell lysate when same amount of total protein (30 ug)

Dear All,

I need to standardize synthesized drug concentration (IC50) for my in vitro experiment but I can't adopt MTT, LDH, or trypan blue kind of assay because I'm working on normal lung epithelial cells and even in my study I need to inhibit the enzyme from the synthesized drug.

Hence kindly suggest a method/ protocol/ idea to determine the drug concentration.

Hi,

I'm working on the immunity of tumor. I injected tumor cells into the mice subcutaneously. After harvesting the tumor, I used percoll and isolated immune cells. Then I added trypan blue and counted cells using a cell counting chamber before flow cytometry staining.

I found in most of the papers, they just said "Count cells for staining". But they didn't say count which kind of cells. Because I saw there were different type of cells in the microscope.

Do I need to count all living cells, no matter how different the shape they are. Or do I just count one specific cell?

Many thanks.

I need to use Trypan Blue solution 0,2% for my research but I can not find a correct formula to do this.

As we are finding difficult to secure the Trypan Blue supplies in Colombia, i would like to know if there is any alternative reagent to Stain the PBMC cells.

Hello,

I am measuring multiple different parameters in cells using fluorescent channels in flow. I would also like to assess well viability. Given that I take the same volume from each well, and do not define a stopping event, can I compare live events between wells as a viability measure? I understand that not all cells that appear live in flow are viable, thus the use of viability stains. Do such stains have to be used to generate viability data from flow?

Thanks!

I need to use Trypan Blue 0,2% for my research, but I can not find a suitable formula to dilute powder.

So I have been trying to standardise a zebrafish single cell suspension for downstream sequencing. I am consistently getting decent viability if I measure it via syto 9 and propidium iodide staining using a commercial single use hemocytometer.

But whenever I try to use the logos Luna cell counter or any other automated cell counter which uses 1:1 trypan blue (0.4%) staining, I am getting really poor results for the same exact samples. Any insight would be much appreciated.

My lab is looking to purchase a cell counter for tissue culture and I was hoping you guys might have recommendations for ones that you loved (or hated)? At a minimum we’d need one capable of counting Trypan blue cells, but ones with fluorescence capabilities might also have use for us.

Thanks in advance for any suggestions you might have!!

Due to the hazard classification of trypan blue we need to replace it with something less harmful and Erythrocin B seem to be a valid option but I find no detailed description on how to use it. E.g. how to dilute the Erythrocin B powder or is it available as a liquid. Has anyone done a validation comparing EB stain vs trypan blue. Is there someone that use Erythrocin B routinely for manual cell counting?

Trypan blue is used to stain dead cells and count them using a heamocytometer. Is it possible to perform this in a 96-well plate similar to the MTT assay? 

Hi! I'm trying to quench fitc fluoresc from Cryptococcus stained with FITC ( at 0,1mM in PBS, 30min, RT) using trypan blue at different concetrations, up to 0,4%.

I'm trying to padronize a phagocytosis experiment by FACS in RAW264.7 mac cell line. First Crypto are stained, washed and co cultured with RAW. The assay looks to differentiate phacocytosed funghi from attached ones by quenching fitc fluoresc from cellls that were not internalized using Trypan blue followed by aquisition by FACS. The point is that , althought it seems to be a something easy, I canot see a substantial efect in fitc quench other than a low reduction in median fluorec intensity compared to not stained cells.

Anyone have an ideia of what might be going on?

tks!

After PBMC isolation and washing, I count the cells of PBMC. Most of the time RBCs exist in the pellet and when I count the total PBMC I am not sure if the counted cells were RBCs or lymphocytes. If RBCs are also stained by Trypan Blue can we distinguish them from other cells to not count them?

Actually I was trying to encapsulate Trypan blue dye in NIPAM microgel for that I mixed certain amount of NIPAM microgel crystals with 10 ml solution of Trypan blue dye solution? stirr for 20 hours and evaporated water at 60 ^oC in oven. Before doing this, major peak of absorbance of dye was around 580 nm but after adding into NIPAM it was shifted to 480 nm. I need your great suggestions. Advance thanks for precious time.

Dear all,

We have performed a classical trypan blue stainning. We would need to decolore it, but by using no toxic products. Do you know if alternatives to chloro hydrate decoloration are existing?

Thank you very much.

I am trying to undergo a cell viability assay with trypan blue staining. I originally got cell suspension by pipetting and observe cells after staining on the hemocytometer.

But I found out my cell seems too fragile after treatment, therefore, I can't see any of the cells under the microscope. (but the cells in the control group are still visible under the microscope, and the cells in the treatment group are still visible in the original wells.)

Thus, I would like to ask if anyone tried to stain cells with trypan blue directly in the well?

Thanks a lot for you answering it.

Hello, I am doing a time course apoptosis assay with AnnexinV following IC60 drug treatment for 24, 48 and 72hr. Through Trypan blue counts we start to see cell death at 48hr. I am seeing a 2X increase in early apoptotic cells in drug treatment relative to DMSO at both 24hr and 48hr. The 48hr timepoint also has a slight increase in late apoptotic cells (1.3X).

At the 72hr timepoint the increase in early apoptotic cells is still present but the late apoptotic cells are the same as the DMSO.

Is it possible that the 72hr timepoint is too long and all the late apoptotic cells are now just debris and thus underestimating the amount of late apoptotic cells?

Hello. So I had seeded some 48-well plates with 70,000 cells/well (human osteoprogenitor) on a calcium phosphate substrate scaffold.

After 24 hours I wanted to study the overall attachment so basically I incubated I removed the scaffolds from the wells and placed in to a new well plate.

I washed with PBS and then incubated in a trypsin 1x solution for 30 minutes to allow for the cells to deattach from the scaffold.

I then neutralized with media and centrifuged the solution to form the smallest dot sized pellet.

I removed the solution and reconstituted in 1 mL of media and then using 10 uL of the solution, mixed it with 10uL of trypan blue.

Transferred 10 uL to side A and side B of a chambress counting slide and used the corresponding automated cell counter by life technologies to count the cells.

I somehow got a bigger number - for example 4.5x10^5 cells alive/ mL

The time point was only 24 hours so it’s not possible that the cells divided that quickly and multiplex in that number so fast.

Can someone please help me understand the principles behind automated cell counting because I believe the machine maybe possibly multiplying by a factor to estimate the number of cells? Please help because clearly the machine won’t count cells which aren’t there, I just don’t understand why it’s spitting out such larger numbers.

Please it’s my last experiment of my thesis and I just need a little help please.

I am trying to grow some Sf9 cells but after incubation at 27 degrees and shaking at 125rpm for 2 days, they all die or at least majority of them are dead.

I grew a 3mL culture (SF900II media) in at 125mL Baffled Erlenmeyer Flasks with initial density of 1.5mil cells/mL (from frozen stock) so after diluting it to 3mL, the cell density becomes 500000 cells/mL. The SF900II media that I used had gentamycin added to it (I added 200uL of 10mg/mL Gentamycin to 1L of media).

Before growing them in the flask as suspension culture, I counted the cells and most of them are alive with some dead. Then, I centrifuged them at 500g for 5mins, removed the supernatant to get rid of the DMSO and resuspended them in 3mL of SF900II medium with gentamycin in it. I then transfer them into a 125mL Baffled Erlenmeyer flask and incubate them in a incubator at 27 degree, shaking at 125rpm for 2 days.

After 2 days, I took 10uL of the cells and added 10uL of Trypan Blue and counted the cells immediately after that. However, most of them were dead (most of them were stained blue) and I am left with around 40000 cells/mL.

Can anyone tell me why is this happening and how I can solve it?

Thank you very much.

I am currently work with antifungal study. I will be doing spore counting by using hemocytometer. I have searched some info that trypan blue can be used to differentiate live and dead cells. However, is this worked on fungal spores too? Any reference or guidelines about this? Is there any other methods can be used for spore counting?

I am working with a herbal extract and currently attempting to determine the appropriate concentration to use for further work, provided it does not cause cell-death- using cellular viability for determining the same.

Unfortunately, my extract is reacting with MTT, leading to the formation of formazon (as evidenced by the colour-change even in the absence of cells). It would be great if any of you could recommend an alternative protocol to check cell-viability (apart from trypan blue assay).

Thanks!

I thawed some MCF-7 cells 3.5 days ago and seeded them into an untreated T75 flask. The medium used was DMEM with 10% FBS, 1% pen/strep, and 0.1% mycozap, all filtered. According to trypan blue assessment by Countess, I had a total of ~20 million cells in 10mL of media with 89% viability at the time of seeding.

By microscopy, I can see that they aren't adhering; cells are still floating around when I move the flask. Is anything suspect about these? Am I seeding too high a concentration?

Hello,

I just started to work with LS180 cells. I used EMEM supplemented with 10% FBS and 1% penicillin-streptomycin to culture them. When I wanted to passage the cells, I realized the majority of the cells have not been attached! So, I decided to re-seed the floating cells in new flask and see whether they are attached or not. After 2 days they were again suspended in the media! To check the viability of floating cells, I used Trypan Blue and counted the percentage of dead cells. As I expected the number of alive cells was less than 50%.

I don't know what is the reason for this happening! These cells are supposed to be adherent, however in my culture, they are not!

Please share with me your opinions if you have experinece.

Many thanks in advance

Assuming I want to passage my MCF-7 cells only once a week with one or two media changes over the rest of the week, does anybody know what concentration (by trypan blue assessment) should I seed into my next passage?

I am using DMEM with 10% FBS, 1% penstrep, 0.1% antimycobacterium, in a 5% CO2 incubator.

Recently, I was trying to evaluate the anti-proliferation activity of a new synthesis compound.

I used the MTT assay to determine the effective concentration at first. Then, I used the effective concentration to perform a trypan blue assay to calculate the number of cells and observed the cell type.

However, the inhibition result of the trypan blue assay is not consistent with the result of the MTT assay.

My question is why did it happen? Is there any other problem I neglected?

I have a sample of Trypan Blue that looks like a tangle of threads in blue and transparent background, but I am not sure if that is what the sample should look like or if it is contaminated and produced something like a fungal hyphae.

I hope you can help me...

Suppose I've given u two samples in which one sample contains live cell and other sample contains dead mammalian cells (Example CHO), Since both the samples are not labelled, I'ld like to know which sample is live cells and which sample is dead cells.

One method i know is :

1) Add trypan blue and observe under microscope, So that live cells appear blue whereas dead cells appear transparent (i.e. no color).

2) Turbidity of live cells increases with time whereas dead cells remain same.

I'ld like to know what all other methods are there to distinguish live cells from dead cells ?

I am having autofluorescence issues that overlap the actual fluorescence from my antibodies. I work on tissue of arteries with atherosclerotic plaques that I fix and store in a solution containing formalin. One solution would be to use brighter fluorophores (Brilliant violet, PE or Texas Red) but is there any way to quench this autofluorescence by using for example trypan blue that I would load on my cells just before flow cytometry analysis? For info, I am currently using AF405, AF488, AF594 and AF680 channels.

Any advice or tip would be welcome,

Thank you !

Hello,

I will use several types of fixations on 8505C thyroid cancer cell line. Those fixations are methanol, ethanol, %4 PFA and few more. My problem here is that how can i know which type of fixation gives me better results. I just want to make sure that my cells are intact and attached the surface of 24 well plate. I will use the materials such as crystaI blue and trypan blue which I already have since I do not want to buy a kit.

Hello. I am currently developing a protocol for a neutrophil phagocytosis assay using flow cytometry. We will use a GFP-expressing bacteria. Can anyone tell me if the addition of trypan blue will quench the GFP-signal of the non-internalized bacteria? I know trypan blue is used to quench the signal of fluorescently labeled extra-cellular antibodies, but I wasn't sure if it would work if the fluorescent signal was coming from protein inside the bacteria?

Hi! I have conjugated a CD34-FITC antibody to 80 nm gold particles. When I stain the cells with the conjugate, I get high rates of dead cells when measuring with trypan blue staining. The ratio of dead cells in the sample ranges between 30%-70% depending on the dilution (the more concentrated the antibody solution, the higher the death rate). Has anyone experienced similar events? How did you solve the issue?

Thank you in advance!

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!

I am planning on assessing cell viability using trypan blue exclusion on some lung fibroblasts. Since these cells have 2 mL of media on top of them, I will have to first collect the media, spin this down, aspirate, and then add trypsinized/detached cells from the plate. Is there a best speed (xg) and time at which to centrifuge the media to pellet the cells (alive and dead) as well as the trypsinized cells if I need to concentrate them more for counting?

I performed a study to determine if carbon dots are cytotoxic on 2 cell lines so I did 2 biologic replicates each one with triplicates of 2 assays MTT and Trypan blue, I applied 4 different concentrations and negative and positive controls.

I don't know which statistical test I should use, two way ANOVA, one way ANOVA, Kruskal Wallis? Neither I know if the data is parametric or not, how can I know that?

Hi everyone,

I just checked sigma website and found Trypan Blue powder got two different versions: 40% and 60% in terms of dye content. When I googled, usually what people say is we just need to weigh 4mg powder and dissolve into 1ml PBS buffer to constitute 0.4% TB solution. I am quite confused as if we have these two different version of TB powder, do we need to take into account the percentage of the dye content within the TB powder? If so, what shall I do to get 0.4% in both case?

Hello,

I have a problem with adhesion of astrocyte C8-D1A line cells. After thawing, the cells are alive (in trypan blue staining, more than 95% viability). But they don't adhese to the bottle. Increasing FBS to 20% did not help. Iculture them on NUNC Delta-surface bottles. I would like to add that after buying from the ATCC collection, they stuck poorly, but grew. I managed to bank in liquid nitrogen and did a number of experiments. The problem appeared after I thawed the ampoules frozen by me. Does anyone have any experience with this? What could be wrong?

Thank you for your help,

Małgorzata

Hello,

I am looking for some expertise on culturing MCF7s. I purchased them from ATCC and I have been following all media and culture conditions. Media is EMEM with 10% FBS and 10ug/mL of insulin. When I plated them post thaw, they had a lot of suspension cells with a few settled colonies. I usually spin down the suspension cells and add back to the flask when I am doing a media change. I have passaged them two times now and I still see the floating suspension clumps. I also did trypan blue staining and there are about 34% live cells in the suspension. I feel it shouldn't take this long to settle down. I have now passaged them twice now and I still see the floating colonies. How long do they take to get to a fully adherent monolayer?

What do these substances stain exactly?

Dear All,

I am growing rice plants in the greenhouse. These plants were inoculated with arbuscular mycorrhizal community with 500 spores/kg dry soil. The rice plant is 45 days old. I took rice root samples for checking the infection of arbuscular mycorrhiza in the root stained by trypan blue 0,01%. When i checked the infection of arbuscular mycorrhiza in the rice root, I recognized these infected forms which was not as I was expected. I would like to know who they were in the pictures. Could you please help me to identify the name of the spores inside the rice root. Thanks you very much in advance.

Yours sincerely,

Xuan Do Thi

I'm pretty sure they are mostly macrophages since after a 2 hour incubation in M199 they are stuck with pseudopodia. My slides just aren't pretty enough for me. I'm using Fisherbrand Wight-Giemsa Stain on rabbit cells from a peritoneal wash (days after drawing monocytes to the location with a peritoneal proteose peptone injection). But my concern is that at some point my counts are off because I can't distinguish between macrophages and other large multi-nucleated cells.

My method:

20uL of 10^6 Trypan blue visualized cells are spread, dried using a Bunsen burner, and then fixed with Methanol for 60s.

Slide is flooded with 1mL Wright-Giemsa Stain and incubated 2 minutes.

Add 1.5mL 1X PBS at pH 7. Gently tipped to mix, for 1 minute.

Stands for 2min.

Rinsed well with De-Ionized water.

Dried and visualized at 400X.

Does it have to air dry rather than use a flame?

I am just having a hard time with the outer membranes. Should I use a different method?

Any help is appreciated!

Hi there,

I'm trying to build a killing curve for HEK-293T towards hygromycin B for the following transfection experiment. I plated the cell at 1.3X10^5 cell/well in the 24 well plates the night before I add the antibiotic. The cell never reached higher than 80% confluence due to the abx until I saw the cell detachment. After 4 days of culture with hygromycin B, the wells that abx concentration was higher than 200ug/ml have cell detached and the cells were floating like a mat in the well. I took the supernatant with the cells and count them with trypan blue but most detached cells were alive. I am a bit confused if I should consider this condition as the cells were killed or they are alive? How do I set the optimal selection abx concentration if this floating cell occurs? Thank you so much for your time!

Best,

Yi

TOTO-3 revealed 'abnormal' nuclei in human fibroblasts from a patient with an alpha-synuclein triplication. Could this be because of an infection or is it possibly because of the cell line? The cells grow without any problems in culture. While counting the cells before setting up experiments (with trypan blue), only a small percentage of the cells was dead. At first sight, the cells look healthy but the nuclear staining suggests otherwise. Does anyone have experience with this?

Hi,

I am working with HMC-1 cells and I want to measure live/dead cell numbers after treatment but for very short time (i.e. 5 minutes up to one hour). This may be tricky since these cells are floating cells and I have an idea on how to perform this study but I have a few questions:

1. seed cells in 48-well plate at 5x10^5 cells/ml

2. let rest few hours

3. add treatment at desired dilutions

4. after each time points, can I take an aliquot of cells (i.e. 10uL) directly out of the well and and mix with trypan blue at 1:1 dilution to count cells? Should I count each replicate separately to get an average of live/dead? I plan on counting using the tC20 automated cell counter, is that reliable for this method?

Does anyone know of a way to stain these cells with trypan blue for example to visualize cell death over short time?

Thank you.

I am working with erythrocytes of mice, to know the effect of Benzo alpha Pyrene on erythrocytes in vitro. Can i perform Trypan Blue exclusion assay to determine the cell viabilities?

I have 50 mL of volume at the beginning of the culture and I just can take samples of 1 mL per day, so there are very few cells to count them using trypan blue (after washing with PBS and detaching with Tryple).

I've tried using Violet Crystal, but it gives me inconsistent results along the days that I've followed the culture.

Does anyone a better protocol, idea or suggestion?

Hello,

I am currently using MRC-5 cell lines and they are at 17th passage. I tried to collect and count them via trypan blue cell counting but I could not see enough cells even though they looked fine in the microscope. I used almost 16 T-25 flasks but couldn't catch enough cells. What could be the problem?

I need to differentiate internalised FitC labeled POS and external surface bound using microscopy on retinal pigment epithelium. I tried quenching with 0.4% trypan blue for 10 min and washing with PBS like a few protocols suggest, but we are not seeing a difference. I understand trypan blue needs to be present to quench the FitC signal, but can't exceed 30 minutes which is not feasible with microscopy. Any help would be appreciated.

I’ve been using a biorad TC10 cell counter and trypan blue to count the cell density.

I wonder if it keeps counting dead cells or not.

For example, if I had 50% viability of 1e6 cells, 5e5 cells are already dead. Then do the dead cells keep affecting counting on next day? or forever?

And is it possible to leave only live cells by spin down? If possible, how many g do you use for that?

BY

Due to the Covid -19 , I'm unable to get supply of chemicals. At the moment, I have only left Trypan Blue with me. Is it possible for me to use Trypan Blue to stain fungus? Please help me with my doubts?

Hello everyone!

I would like to ask you, if it's possible to count viable cells before we freeze them (in PBS, isolated cells from lymph nodes etc.) using flow cytometry instead of manual counting with Hemocytometer ( Neubauer chamber ) and marking cells with Trypan Blue?

Thank you in advance.

usually, Trypan blue stain is applied before seeding cells in 96 wells plate and cells are count by hemocytometer. I am confused about how cells will be counted in 96 wells plate by trypan blue stain after drugs or inhibitor treatment.

I am using U251 Astrocyte for my cell culture and I don't know the passage number of the source when I received it.

Now I passaged them many times and I want to check that they are in a good situation or not.

I know that flow cytometry can help a lot, but which type of the assay is good for me?

I count my cells by Trypan blue and countess machine life technologies and I know the percentage of live cells and dead cells, but I don't know using viability assay by flow cytometry can help or not?

Or Do I use cell cycle assay by dying PI or cell proliferation?

How can I found about my cells situation that they are in good situation for doing next experiments.

Our lab uses a histopaque density gradient to isolate the PBMCs from whole blood. Once the cells have been isolated and are ready to be washed, I have noticed that our cells adhere very strongly to the bottom of the tube. I cannot flick the bottom of the tube to remove the PBMCs. Typically, I have to use a disposable pipette to gently resuspend the cells; however, I am worried that I am lysing the cells thus lowering their viability.

Does anyone have any suggestions as to why this may be occuring?

Also, we typically assess viability using a 1:1 trypan blue stain with the Countess II. We have noticed that our viability is not very high and is often variable. Other labs in our department use the PBMC isolation tubes and achieve similar viability when using the same protocol (i.e., 1:1 trypan), so this leads me to believe that it is not our protocol. The viability is also >90% when counting with a hemocytometer.

Has anyone had success counting using the Countess II and a trypan blue stain?

Can I pipette cells in each well and then mix 10 uL of cell with 10 uL of trypan blue?

Hello,

I work with 21-day differentiated Caco-2 cells and I want to do a trypan blue count.

Unlike undifferentiated cells, trypsin (i use TrypLE Express to be exact) does not allow me to separate cells from each other and I get very large cell clusters.

What could I use to separate them effectively without inducing rapid cell death?

Thanks in advance

I recently started a U937 line. On the day I thawed the vial of cells they were at 50 % viability (by trypan blue exclusion). When I checked them the next day they were at 10 % viability. Has anyone else observed this large drop-off in the first 24 hours post-thaw? The original vial was from the manufacturer so I don't think the cryoprotectant was the issue. Thanks in advance.

When freeze thawed working bank of Human Fetal Fibroblast (Dermal) were cultured with a specific seeding density, the yield on day 4 was similar to that of seeding density. When all the working banks of the same batch were tested for their yield, almost every bank gave similar outputs (except two banks). The culture medium was evaluated for composition check and cells for mycoplasma contamination. Medium was found appropriate and cells had no contamination. The incubation conditions were also checked. Even the pipettes and hemocytometer was calibrated and standardized with qualified cells prior to each run. All the cells were viable during counting (Trypan blue). The trypsinization protocol was 100% efficient during each run. The culture flasks T25/ T75 of three different companies were included to verify flask variation (if any). The liquid Nitrogen dewars were constantly monitored for liquid nitrogen levels.

Still the HFF cells yielded the same seeding density on day 4. These cells remain viable but refuse to multiply. Kindly give suggestions.

Hello,

I am isolating primary murine glial cells from different neonatal knockout strains (p0-p2). After several rounds (up to 4 times) of harvesting the microglia, I am dissociating the astrocyte monolayer from PDL coated 75 cm² flasks (containing 2 brains). At this step, I am having issues getting single cells, the astrocytes are sticking together and forming big chunks of cells.

Although I have aleady tried different methods of dissociation such as using different kinds of narrow pipettes, trypsine, DNAse, papain, cell strainers and so on, I am still getting big chunks of cells. The cells seem to be fine since live/dead staining with trypan blue indicates that the cells are pretty viable (less than 5% dead cells).

Does anyone have a clue what makes the astrocytes stick together after their dissocation from the cell flasks?

Thanks in advance!

Kind regards,

Laura

I am working with J774A.1 cells purchased from ATCC on 1/16/18. I have worked with many different cell lines, but this one is turning out to be a little trickier for me. I'm looking for advice/feedback/anyone's thoughts or help!!!

Issue # 1: the cell morphology appears to differ from anything I've worked with. Some of the cells appear normal and unactivated, while others appear activated, despite the lack of any endotoxins or contamination. (See attached image.) The cells are growing in Hyclone DMEM supplemented with 10% FBS and 1% Pen/strep/L-glutamine solution ( L-glutamine 200 mM, streptomycin 10 mg/mL, penicillin 10,000 units.) The cells were thawed upon arrival and plated in pre-warmed media defined above and placed in a humidified incubator at 37 degrees C and 5% carbon dioxide. After the cells were allowed to adhere for 6 hours, the initial media was aspirated and replaced with fresh, pre-warmed media. The cells were split to a 1:6 ratio after reaching 70% confluency. Do these cells look normal? What is with the variations in morphology, and can anyone explain what is going on in the circled cells in the attach image?

Issue # 2: I am having a great deal of difficulty counting the cells for seeding plates. These cell membranes appear to be much more sensitive to scraping, which is throwing off my cell counts via trypan blue using a hemacytometer by approximately 60%. Because we use these cells for a variety of applications in our teaching labs, I need to figure out the best way to detach cells without damaging the cytoplasm so much that the trypan blue leaks into the live cells. We want to avoid using trypsin. Any suggestions???

Many thanks!

Hello, for a current microscope system we use, only bright field imaging is available, not phase or DIC. Is it possible to add a dye to cells to increase their contrast for bright field imaging? Something like trypan blue, but for live cells? Thanks.

I want to observe fungal structure under the microscope. As trypan blue staining need to use expensive chloral hydrate...., I want to try another method... Can anyone introduce me an alternative staining methods for fungal structure observation in the infected plant tissues (leaves)?

Thank you very much~!!!

Dead cells or cell debris are supposedly to be lighter than live cells, and therefore the most recommended procedure is to centrifuge and remove supernatants.

Recently I used centrifugation at 200g for 3min to pellet live cells, but found that 90% of the cells showed dead by trypan blue staining.

I also read other's recommendation on internet and collected medium without disturbing dead cells/cell debris on the bottom of culture dish, but still got the same result.

This is most frustrating....

Somehow I felt confused:

if the dead cells actually sink to the bottom while live cells remain in suspension, how could centrifugation separate them by pelleting live ones?

Or maybe I could stall the cells in falcon tube and wait for debris to fall down with gravity?

I have a question about growing dinoflagellates in IMK seawater medium. I count them by staining 1:1 with a 0.4% Trypan Blue solution. I have checked that the Trypan Blue and the medium itself are not full of clumps. But the Trypan Blue stains large blue clumps in the culture medium. Is it possible that the dinoflagellates are releasing a protein into the medium?

(This is a non-axenic culture, but the clumps are not living.)

I am currently performing an HPRT mutation assay by which cells are treated with a mutagen and then plated in the presence of 6-thioguanine such that only cells with HPRT mutations will survive and form colonies.

What I am noticing, however, is that now that I have plated the cells in 6-TG containing media, it is difficult to tell which cells are alive and forming colonies and which are dead. I see many clumps of cells that look like colonies, but there are too many of them for these all to be live colonies (I have tested the 6-TG concentration I am using on these cells and it should be killing all normal cells). A protocol I read also said that cells may not be immediately killed by 6-TG, so they may have started proliferating and then died.

We normally stain colonies with crystal violet, which works well when cells are at a low density and colonies clearly stand out, but at the high plating density needed for this assay I can see that it will be very difficult to tell where the real colonies are if I do crystal violet staining.

The only mention of this I see in protocols for the HPRT assay is one protocol which says that trypan blue staining and "morphology" can be used to distinguish dead cells. I have found a couple protocols for trypan blue staining and fixing of adherent cells in dishes, so I think that should work. However, I do not think I could distinguish between crystal violet and trypan blue if I use both, so I am not sure if that is an option.

I was hoping there was a way I could stain the colonies with both trypan blue to mark dead cells and another dye to mark live colonies (or a different combination of dyes altogether). I would like to be able to visually count and mark colonies as I do with crystal violet staining given that I have 40 15-cm plates of cells and will be doing many replicates of this experiment. Is there any way I can do this?

EDIT: I think I have identified what it was about the assay conditions that was leading to these ambiguous results. I will update this question once confirmed.

Hi everyone,

I'm looking for a relatively simple viability test method to distinguish between live and dead spores (fungi i.e. Botrytis or Penicillium). I need to be able to do this using a compound/optical/light microscope and a counting chamber. I was looking at trypan blue but the warning labels (carcinogen) diverted my attention to methylene blue. Can anyone give me some background on these or other test methods? For now, I want to try and avoid expensive equipment (fluorescent microscopes and flow cytometry). Any suggestions to alternative systems that might be cheaper?

Thank you,

Pieter

I have been trying to thaw out lines of DISC1 cells of the same batch of cells onto 12 well plates (frozen down a month and a half ago). However on doing a Trypan Blue assay it seems to be that even though the cells are viable at the time of thawing (.13 million cells/ ml) they seem to all die after being plated out. Can't be an issue with Matrigel coating as XCL1 lines successfully adhere to the plates while using the same batch of Matrigel. Do let me know if you have any suggestions as to how I could tweak the standard thawing process with these slightly sensitive cells.

I'm developing an assay to determine sensitivity to a hypomethylation chemotherapy agent. Preliminary cytotoxicity assays were conducted to determine dosages that yield the optimum viable cultured cell count to hypomethylation ratio via Trypan Blue & Cell TitierGLO assays. In the attached file you can see i have graphed the data with error bars. Is it necessary to conduct any more analyses via using methods such as ANOVAs?