Organelles - Science topic

There's no staining protocol, the cells I use express a GFP that allows purification with anti-GFP dynabeads. Basically I incubate a membrane fraction with the dynabeads and after the wash steps I put fiew µL of resuspended beads and place them between slide and coverslip. The dynabeads (2.8µm) are the spheres that you can see in BF and are slightly autofluorescent. The images are from Wild Type cells, the contaminant indicated by a red arrow is not related to GFP. In my conditions of interest I capture my GFP-vesicles on the surface of the beads and they are distinguishable from these contaminants.

Me too..

Helena Milionis Do 25 uL of lysosomal eluate + 50 uL of substrate, incubate 30 min at least, then stop with 200 uL stop solution. This usually works for magnetically-purified lysosomes, but you can prolong the incubation time for sure. In some cases (exocytosis assays) I've increased it up to 24 hours.

Hi Cécilia,

Did you solve the problem you shared? I am also working with HA antibody and MitoTracker in my research and I am experiencing a similar problem. I used FBS and BSA in buffers. I'm waiting for your experiences.

Best wishes.

It would be useful if you provided a picture and explained how you labelled the nucleus and protein of interest and what sort of microscope you used to take the image etc. Qualitative analysis of protein localisation is fairly easy but quantitative analysis depends so much on how your sample is prepared for imaging and how you take your images. A lot depends on the question you want to answer as well. For example you can get numbers from software such as ImageJ (FIJI) (which is free) but you need to know what to measure.

Dear Dr. Shilpa Patil

Ferroptosis is a type of iron-dependent regulated cell death mainly caused by unrestricted lipid peroxidation and subsequent membrane damage. Selected metabolic changes, such as iron accumulation and lipid peroxidation, are considered as the biochemical hallmarks of ferroptosis. Different organelles are involved in this metabolic cascade, which eventually leads to the rupture of the plasma membrane. So, in addition to mitochondria and ER, the other organelles involved in ferroptosis are lysosomes, peroxisomes, Golgi apparatus, lipid droplets and the nucleus.

You may want to refer to the article attached below for more information.

Regards,

Malcolm Nobre

You have to calculate the ratio of fluorescence emission intensities at 2 different excitation wavelengths for each organelle. Convert this ratio to pH values by using standard curves for the ratio of fluorescence emission intensities versus pH.

Ex. for Lysosensor Blue-Yellow, calculate the ratio of fluorescence emission intensities at excitation wavelengths of 450 nm and 405 nm (F450/405) for each organelle. Then, convert these ratios to pH values by using standard curves for F450/405 versus pH.

Warburg effect or aerobic glycolysis cannot be viewed from an energetic point of view. It requires a multimodal approach.

Lets start with a contrafactual analysis. What would happen if a malignant cell would not use aerobic glycolysis.

Proliferation reduces oxygen availability because angiogenesis is quite ineffective to compensate for the increased need of oxygen.

In second place, the production of reactive oxygen species would induce a very dangerous oxidative stress. Warburg effect prevents both.

The main purpose of the Warburg effect is not to provide energy to the cell but to create building blocks for synthesis of amino acids, purine and pyrimidine precursors, and antioxidative molecules.

The energy imbalance is compensated by two mechanisms:

1) Increase of the glycolytic flux with a glucose uptake that increases 10-17 folds.

2) The oxidative metabolism may be downregulated but it is not eliminated.

Furthermore, during Warburg effect, almost 50% of glucose can be metabolized through the OXPHOS pathway.

Tumor cells need to keep the Krebs cycle working.

Therefore, the idea that malignant cells use the glycolytic pathway exclusively without OXPHOS is a well established mistake since the Warburg days.

The LysoTracker probe is typically designed to work best in the acidic pH range found within cellular lysosomes. These compartments in cells have a pH range of around 4.5 to 5.5. The probe is specifically designed to accumulate in these acidic compartments, such as lysosomes, where it can emit fluorescence and allow for visualizing these structures.

It's important to keep in mind that the exact pH range and performance characteristics may vary depending on the specific formulation and version of the LysoTracker probe you're using. Different versions may have slightly different optimal pH ranges and specifications tailored for specific applications. To get the most accurate information about the working pH range and optimal conditions for your specific LysoTracker probe, I recommend referring to the product specifications or consulting the manufacturer's instructions.

Hello Ziqi Liu

Please refer to the article attached below. It may be helpful.

Best.

Can you clarify what you mean by microbes and organelles? A common standard definition of microbes is prokaryotes and archaea, neither of which typically have organelles.

If it was (living) yeast, they'd definetly be dividing in a normal media and you would notice turbidity in the medium rather sooner than later. If you suspect any sort of contamination you should not use the culture media in an incubator with other experiments or use as a component in eg freezing media, that might be handed around in the lab. Also clean the incubator thoroughly after testing the media. The staining suggestions from Can and Tomás above seem indicated, a trypan blue staining will give you some additional insights and an occasional test for mycoplasma is not wrong when working in labs that are at risk. I would expect an occasional dead cell in a medium if somebody pipetted carelessly, but if they are occuring often, somebody would have needed to empty a lot of old supernatant medium into the bottle, or the cells would have died there after growing there for long enough to induce the colour change. However, most contaminants are sadly not that picky on where they grow and I expect you'd add Pen/Strep or similar right after opening a bottle and not after things already started growing. In general, if the medium has changed color, I would not recommend using it any further (even if cells would still grow in it), because the change in pH might introduce unnessecary variation into your experiments. Some other considerations: are you sure these come from the bottle with medium and are not present eg in you culture plates, pipettes or any other equipment? especially if you have filtered the medium, I would not expect a lot of debris. Have you opened the bottle freshly and has it been stored correctly? Did you have added any supplements to the medium (like FBS) that could have introduced these? If you are in a lab where people collect supernatant for various purposes, check with your colleagues whether this is an unlabeled case of collected supernatant. If the bottle has been opened freshly by you, I would check with the manufacturer for additional insights, as they know how the components behave under different storage conditions and usually reply rather fast.

Yes it worked very nicely, I have used Mito Tracker Green, LysoTracker red of Thermo Fisher Scientific for fixed cells,

and I am sure ER tracker blue will also retain after fixing the cells

Ravindra Thakkar Prashant Pandey Thank you for your help. I found a similar structure in the following paper and I think it should be lipid droplets or lipidic granules.

Hello Kaneez Fatima

Immunofluorescence co-localization would be your easiest best choice.

Good Luck

Lina

Yes, you are right. Paraformaldehyde fixation can lead to the dissociation of protein-protein or protein-nucleic acid interactions in the cell cytoplasm as well as in sub-cellular organelles and lead to misinterpretation of the actual localization within a cell. For more detail see the link given below:

Paraformaldehyde Fixation May Lead to Misinterpretation of the Subcellular Localization of Plant High Mobility Group Box Proteins. PLoS ONE 10(8): e0135033. https://doi.org/10.1371/journal.pone.0135033

Basically three staining solution used to observe cell organelles under microscope are methylene blue, neutral red and safranin. These stains are essential for observation of cell organelles. Cells are quite small and transparent Gaurav Kumar

Dear Abdul,

this is a very interesting technical question. We did only very little work with ruthenium complexes in the past. However, I think that the main advantages of ruthium complexes are that (a) ruthenium has different stable oxidation states, (b) most ruthenium complexes are kinetically stable (i.e. they don't easily decompose in solution), and (c) through the use of specially designed ligands they can be made water-soluble so that they can be utilized under physiological conditions. Various ruthenium complexes are now well established in the diagnosis and treatment of cancer. For a good overview about this topic please have a look at the following very interesting review article:

Fortunately this review has been posted by the authors as public full text on RG. Thus you can freely download it as pdf file.

Good luck with your research work!

Oops!

the paper!

Thank you for your suggestion Maurice Ekpenyong . Although I’m hoping to look at enzyme activity rather than any cytochemistry. Reading the paper, it sounds like DMSO and snap freezing with LN2 similar to Freezing live cells would be enough. I may have to run a comparison.

best wishes.

I think this may help you

We couldnt really get Golgi and ER separated but the separation we got was sufficient for our purposes. Its been a while since I did these experiments but we published the results here:

There you see how "good" our seperation was. I hope that helps. Good Luck

Hi usually we do imaging a day after the transfection because sometimes after the transfection if you leave it for 3 days. The non transfected cells can over grown on top of the transfected cells which would then be on your way. So ideally we do imaging the day after transfection.

Hi,

Have you solved this problem? I have the same problem with MEF cells. I used mitotracker to stain cells. Mitochondria in all other cells look tubular or branched. But in many MEF cells, they are quite fragmented.

Best regard

Yuan-Yeu Yau wow! Is it true Wenlin Liao ? maybe if you upload your own photograph we may help you better ;-)

Thanks!

Since there are a lot of steps in preparing liver tissue for TEM, it is difficult to say where you might be going wrong without more information about the protocol you are using.

I would first check that you are using the proper fixative. I use 2.5% glutaraldehyde buffered with a cacodylate-sucrose solution (0.1 mol L-1 sodium cacodylate and 0.1 mol L-1 sucrose, pH 7.6) and then fix at 4°C.

For the processing, I stain with 1% OsO4 first and then with 0.5% uranyl acetate followed by ethanol dehydration and embedding. There are wash steps in between of course, I use 0.1N acetate buffer. This protocol works well for me but there are other very good ones out there too. If you are able to find this book, then I recommend it highly: https://www.springer.com/gp/book/9780306477492

- Melissa

Hi Lalit,

In my opinion, Rubisco is the best candidate for your experimental setup. Functional Rubisco consist of only two subunits (large and small subunits). The small subunit is encoded by the nuclear genome while the large subunit by the chloroplastic genome. It will be better if you use both of these as a negative and positive control. The large subunit will provide you the quantity of chloroplastic transcript while the small subunit will suggest the purity of isolation.

Best

vivek

Also, see the link below.

Thanks for the answers! I want to clarify that I am interested in extracting phospholipids, not proteins. Thanks!

Some organelles like the Golgi complex undergoes fragmentation during mitosis and then reassembled after mitosis. The process is very extensively studied.

Hi there,

I also have never done this, but I am afraid that the preparation of the mitochondria will introduce artifacts and you will not learn much from the staining.

However, I am wondering, what you could do to efficiently get the mitochondria onto your slides. Maybe, CytoSpin would be helpful.

You probably need some highly adhesive slides to keep the mitochondria stable. I think, SuperFrost Plus adhesion slides would make sense.

Furthermore, I would try fixation with 4% PFA for approx. 5-10 min, since you don't need much diffusion if you only have isolated mitochondria. I looked into the composition of MiR05, the PFA will probably react with the taurin and BSA, but it does not matter, since PFA concentration is high enough and you don't need these substances for the mitochondria after fixation...

Both, Tween 20 and Triton-X100 are non-ionic detergents, but I would agree that Tween 20 is probably a bit milder than Triton-X-100.

Good luck,

Sebastian

Hi,

Please find the attached link...

See: Cell Calcium 2008 Oct;44(4):386-99.doi: 0.1016/j.ceca.2008.02.002. A new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum

@Tho Ho I got avobe answers.

Dear ,

Witht the danger to make things even more complicated…unfortunately. See for examples of excellent overviews of what we are talking about:

Bernardino de la Serna, J., Schütz, G. J., Eggeling, C., & Cebecauer, M. (2016). There is no simple model of the plasma membrane organization. Frontiers in cell and developmental biology, 4, 106.

and

Ingólfsson, H. I., Melo, M. N., Van Eerden, F. J., Arnarez, C., Lopez, C. A., Wassenaar, T. A., ... & Marrink, S. J. (2014). Lipid organization of the plasma membrane. Journal of the american chemical society, 136(41), 14554-14559.

In conclusion it very much depends on what you want...

I think one of the best protocols is described in (see enclosed file):

where you (hopefully) recognize the various steps I talked about in my previous answer.

Again I hope this heps you a bit further.

Best regards.

No problem Fernando Ángel Fernández-Álvarez . I found this today :https://github.com/ndierckx/NOVOPlasty/wiki/Interpretation-&-post-processing

Hope it helps!

Martin Klvana just fine, okay it wouldn't hurt to read this article The Theory of Everything It is based on duality.

https://www.academia.edu/38670214/The_Theory_of_Everything

Dear Alicia Virseda Jerez,

thank you for the reference. I have found the paper, and, as I have expected, the authors used culture medium with Pen/Strep. This is a very good demonstration of why I recommended not to use JC1 at all. The problem with this die is that it sticks to anything. In cells grown in the presence of streptomycin mitochondria do not function normally. They are alive, they will respond to changing situation in the cell, but they do not respire, they have no oxidative phosphorylation. I have many times different cells grown without and with Pen/Strep, and in all cases isolated mitochondria had no respiration. This is a worldwide tragedy, that people working with cells use Pen/Strep and think they do Science. Douglas C. Wallace, the Father of mitochondrial Medicine, every week used to warn us do not use antibiotics! Mitochondria are former bacteria< therefore, they are sensitive to antibiotics. I (that is Dug) instantly fire anyone, who uses antibiotics. I was lucky that I studied cell culturing in his lab. My warned my friends, who use cell culturing< said to me that after they stopped using Pen/Strep, they started to obtain absolutely different results that make sense. Do not waste your time and Life doing nonsense.

Late to the party but I have performed sub-cellular fractionation on frozen heart tissue with good results - yet to be published. This paper describes liver tissue frozen and subjected to fractionation also with good results.

Just give it a try, most of the important nuclear proteins complex with a structural binding partner (DNA, Lamins) anyways, so will be probably remain in the nuclear fraction even if the membrane has been a bit pierced with ice crystals. also, careful flash freezing i.e. in isopentane, should limit the number of crystals that form.

Hi

open this link maybe it will benefit.

Perhaps easiest is to spot-blot the fractions (1-2 µL) onto a PVDF-membrane and then develop just like a western blot.

Usually with a CMV driven vector transiently transfected in to a cell line you will get a huge range of fusion protein expression levels (usually beyond the dynamic range of a single image with most imaging platforms). The expression level will depend on the fusion protein half life, the time after transfection, - or more accurately nuclear access- and the number of copies of the vector introduced into cells. Cells will only start to express protein from the vector after mitosis and the levels will then tend to increase untill the cells are fixed.

Even if your endogeneous protein is cell cycle regulated in abundance then you should still get a good linear relationship at the top end of the expression levels, the regression line will just not go through zero. The non-transfected cells in your population will give you a good idea of the dynamic range of the cell cycle regulated endogenous protein levels. Incidently, after a transient transfection (say after 24hs with fugene6 ) transfected cells usually appear in pairs with similar expression levels. The distance between the pairs of cells can give you a crude estimate of the time since mitosis.

Phospho specific antibodies are more of a challenge - you could try expressing a phospho-mimetic mutant vector to see if that works with your antibody, if it is specific for a particular phosphorylation site?

We once managed to get a really good polyclonal phospho specific antibody to a serine in I kappa B alpha that on western blots was much more sensitive (at least 10x ) than the antibody to I kappa B alpha itself, which just goes to show how varied antibody affinities are.

Super nutrition - You mean Food Suppliment like Vitamines / Minerals / anti-oxidants .

Thanks for all .... osmosis process occurs in bacterial and other cells.

But is it possible to virus?

I have never used DiD dyes before, but quick research gives some clue to your problem. Here is the paper on PMC "PMCID: PMC5101988".

And I quote "The staining of live cells enables the observation and separation of cells following direct co-culture to analyse any alterations in the phenotypes of co-cultured cells. The application of lipophilic cell labelling dyes from the DiO-family is a powerful tool used for the staining of live cells. These lipophilic fluorescent carbocyanine dyes have been used since the 1980s in vitro and in vivo due to their high quantum efficiency, the simplicity of staining protocols and reduced cytotoxicity compared with hydrophilic dyes (1,2). The excitation and emission spectra of lipophilic fluorescent dyes enables multiple staining (2). Dyes from the DiO-family label the plasma membrane and migrate to cellular organelles as a consequence of membrane turnover (3)."

So it appears what you observed is a normal phenomenon.

It really depends on what your specific needs are for the work that you are doing. For basic cell culture, refrigeration is not needed. If on a rare occasion you do, then put the centrifuge in a cold-room. Either rotor type is fine. Any cheap lab centrifuge will have a sufficient speed range.

You can use qPCR based methods. The main problem is that you need to pick up the appropriate primers for single-copy regions in nuclear, chloroplast, mitochondrial DNA. Here is the article (DOI 10.1007/s00294-015-0482-1) with a similar method.

Sucrose step gradient ultracentrifugation is a good way to purify plasma membrane away from other membranes. When I used to do this with CHO cells, the plasma membrane was the lightest fraction and was isolated from the 16%/31% sucrose interface.

Engelbert Buxbaum

Thank you for your reply.

I think we will try different concentration of non-ionic detergent first. Nitrogen cavitation seems nice for studying localizations of protein interactions; however, we don't have the tools for that.

And, after a second thought, it sees like using in vitro liposome with recombinant protein to identify the effect of specific lipid compositions on membrane association of protein might be better (e.g. DOI: 10.1126/science.1092571 Fig.2B)

Once again, thank you for your comments.

thank you Sudharsan Kannan!

Apicoplast apparently is not involved in cellular invasion - perhaps you misread Wikipedia article. It synthesises fats and isoprenoids for cellular metabolism. Those that are actually involved in invasion are apical secretory organelles including micronemes and rhoptries. If you look for the molecular mechanism of invasion, merozoite surface proteins is the answer.

Manoj Chandran it is a good question, I think that plantoids are a good example of this application in real life. Although prototypes are still in development, the concept itself is interesting as energy-generating models.

Professor Stefano Mancuso has a very good TEDtalk who talks a little about it: https://www.ted.com/talks/stefano_mancuso_the_roots_of_plant_intelligence

For volume, I think you are OK. If you want to use intensity for comparison of abundance by mean or integrated intensity, then you cannot have saturated pixels.

Dear AKS, it is a mind boggling question and lot of overlaps exists. Before we discuss we need to under physiological plant disorders which are mostly caused by environmental or say abiotic factors ( low light, weather vagaries, water-logging or nutrients deficiency) affect the functioning of the system. The symptoms of physiological disorders can be prevented by altering environmental conditions. However, once a plant shows symptoms of nutrient deficiency it is likely that that yields will be affected. We can say that the implications are any kind of abnormality in economically important part of plant or other parts that contribute to yield and quality of vegetable is termed as physiological disorder.

You can quantify using DLS for the size of exosoma and Nanodrop for the absobace at 280 nm to define the number of particles . I use a scale made with protein BSA nanoparticles.

I am anxiously awaiting these results.

Hi

things that you can try

1) not use the sorter. Limiting dillution: plate 0,25 cells per well. This should lead to clonal cell line

if you want to use the sorter

2) use 50% conditioned medium in the initial,step of single cell cloning.

3) during the sort use a larger nozzle (and low speed...). This will reduce the stress on the cells

4) include 5 and 10 cells per well. based on the number of clones that grow out you can statiscally say whether they are clonal.

But the medium was clear and if you look at the atcc site on the photo of this cell line specification they also have quite visible dark spots. Are you sure about bactetial contamination ?

Mitochondrial size and shape depends on a variety of factors, especially in cultured cells. Hence all these "observations" are relative to the conditions/methods employed and relative to appropriate controls and likely differ more or less from in-vivo behavior.

Both methods have been shown to possibly lead to cellular stress that may manifest itself in changes in the mitochondrial network (especially in live cell imaging).

I do not think you can draw the conclusions you stated without additional evidence (in your case co-labeling fusion and fission machinery etc.).

Useful draft for you

Best wishes

Hi Suraiya,

Thank you very much for the information. I'll try to figure out how to modify these approaches to assay organelle-organelle membrane exchange.

Do you need a protein? There are lots of small-molecule PS II inhibitors, DCMU, atrazine, etc., that inhibit PS II function but do not affect other parts of the cell unless at excess concentration.

The word you are looking for is "dynamic range". The dynamic range in your images seems to be too small to see the weak signal (peroxisome) without saturating your strong signal (plastid). If I understood you correctly.

There are several ways you can try to improve on this. First, what bit-depth are you using, usually you can set the readout from the PMT to 8 or 12 bit, 16 bit on some systems. While this not increase the dynamic range as such, it subdivides it into more steps (256 for 8 bit, 4096 steps for 12 bit). Higher bit will make the files larger and might affect acquisition speed, but might give you more details on your weak structures.

Another option is HDRi (high dynamic range imaging), which you might know from your phone. In principle this can also be applied to microscopy images. A series of exposures is taken with different settings and then combined into a single image by software. Some microscope software has this function integrated but it can be done externally (e.g. using imageJ). However, be aware that this is a non-linear operation and the resulting image will not be quantitative anymore.

Here are a few articles on the mechanical properties of cells.

Regards,

Joachim

The culture technique are usually regarded as the gold standard method for

detection of Mycoplasma as it uses a selective growth media and incubation conditions to

grow any Mycoplasma present in the sample. Most Mycoplasma contaminates can be

detected on standardized agar like SP4 media or PPLO broth with subculture on PPLO

agar with the exception of M.hyorhinis. Colonies of Mycoplasma exibit a FRIED EGG

morphology when viewed under the plane microscope. For a distinctive result at least

incubation on an average of 28days is required

Figure 13: Colony of Acholeplasma laidlawii showing typical "fried egg"

morphology.

First, your image is rather substantially out of focus: the hemocytometer gridlines should be sharper. The illumination is also poor, so contrast is low. If you improve your imaging, it will be easier for you to do your counts.

As Dr. Stefansson notes, this is an exclusion assay, so viable cells will exclude the dye and will remain clear or white. Any sign of blue within the cell indicates a compromised cell membrane and thus those cells should be counted as non-viable.

What are your staining conditions? I use the classic method from the Gibco Cell Culture Basics:

0.4% trypan mixed 1:1 with cell suspension.

That book dates back to 1999 and the semantic is now obsolete.

The second chapter of this review clarifies the topic.

Best Regards

Dear Alex, thank you for the answer. It is very helpful. Have you tried your protocol for nuclei extraction not from cell culture but organ?

Dear Wolfgang,

Many thanks for your interest. The textbook is a general biology textbook, and I am only in charge of the "cellular biology" an "membranes" chapters. So this needs to stay simple, as it is aimed at first year students only. Thus, I need "normal" cells examples. I do not need semithin resin sections because I am not in charge of the hystology and physiology chapters.

Here is a list of the images I still need to find (TEM images mostly):

-High resolution plasma membrane cross section, showing the lipid bilayer

-Endocytosis or exocytosis figures that I can assemble to illustrate transcytosis

-One beautiful representative animal cell with most main organelles

-Budding yeast (SEM or TEM)

-gap junctions

-adherens junctions

-smooth endoplasmic reticulum

-Nucleus with nucleolus, hetero and euchromatin

-lysosomes

Thank again for your answer, I hope this clarifies my demand.

Hey Dan!

The "endogenous control" you're suggesting would likely be the most elegant approach but may be tough to find.  

As an alternative, you could think of disrupting the assembly of P-bodies through more than a single "exogenous" approach.

Check out the link to an RNA biology paper describing a screen for myxobacterial compounds which inhibit P-body assembly.  Gephyronic acid, a protein synthesis inhibitor, efficiently blocks P-body assembly. In addition to a pharmacological approach, you could envision a DDX6 siRNA. 

These controls may be weaker than a cell line naturally unable to assemble P-bodies, if it exists, but providing two different perspectives may make the conclusions somewhat stronger. 

Dear samrina Ahmed ,

You can get full of information about different types of mechanism in plant cell which involve in generation of antioxidants. I suggest to read the book entitled "Antioxidants and Reactive

Oxygen Species in Plants" edited by NICHOLAS SMIRNOFF,

School of Biological and Chemical Sciences,University of Exeter,UK. You can see from the link below.

http://onlinelibrary.wiley.com/book/10.1002/9780470988565 

Hi, Dear Jindou Dong

Regarding your question (How to quantify dynamic loose organelles in cells?), please check the following, I hope useful,

Best Regards,

www.springer.com/cda/content/document/cda.../9780387897806-c1.pdf?SGWID...

hi

this shapes seems as yeast ( contamination from soil or other source in your lab ...) .

good luck

You can encapsulate a fluorescent dye in carrier and perform suitable imaging to visualize whether fluorescence occurs outside the cell or inside it. 

Certainly, mitochondria K-cycle and respiration (K-O2 transport)  the key function in human living cells.