In Vitro Culture - Science topic

Thank you very much for this protocol, have a good day

In diabetic wounds, the levels of growth factors (GFs), such as platelet-derived growth factor (PDGF), are generally reduced, leading to impaired wound healing. The chronic hyperglycemic environment in diabetes impairs cellular responses, including the production, release, and activity of growth factors, which are crucial for tissue repair and regeneration.

Can Growth Factors Be Imbalanced in Diabetic Wounds?

While the reduction in growth factor levels is commonly observed, imbalances, including both decreased and increased levels, can theoretically occur depending on the stage of the wound and the body’s response to the injury. However, sustained elevations in growth factor levels are unlikely because diabetes disrupts the normal regulation of growth factors, and any temporary spike in GF levels would still likely be inadequate for effective wound healing.

Some studies suggest that growth factor therapy (e.g., recombinant PDGF) can help boost wound healing in diabetic patients by compensating for the deficiency, but this requires controlled delivery to be effective (Sen et al., 2009).

References:

Hai Ping Yu

i have some trouble with my neutral red assay. I get highly variable values in my cell free controls (only medium) and i think it has to do with precipitation of the dye. Did you have any luck with your trouble shooting? Help would be highly appreciated :)

You could try a different basal medium. Woody Plant Medium (WPM) might be a good option. Try a different range and combination of your two hormones (eg. high IBA with low NAA, moderate of each, low IBA high NAA).

Sure! IEC6 is a commonly used cell line in biomedical research, particularly in the field of gastrointestinal physiology and pathology. The IEC6 cell line was derived from normal rat small intestine epithelium. It was originally isolated and characterized by Quaroni and Isselbacher in 1982.

IEC6 cells have been extensively studied due to their ability to proliferate rapidly and form monolayers in culture, making them a valuable tool for investigating various aspects of intestinal cell biology, including epithelial barrier function, nutrient absorption, and mucosal immunity.

These cells retain many characteristics of normal intestinal epithelial cells, including the expression of markers such as villin and sucrase-isomaltase, which are indicative of differentiation into absorptive enterocytes. Additionally, IEC6 cells have been shown to express various transporters and receptors involved in nutrient uptake and signaling pathways relevant to intestinal function.

Researchers commonly use IEC6 cells to study intestinal physiology, epithelial barrier integrity, responses to pathogens and toxins, as well as to investigate the effects of dietary compounds and pharmaceutical agents on intestinal health. Furthermore, the ability to culture IEC6 cells in vitro provides a convenient model system for studying molecular mechanisms underlying gastrointestinal diseases such as inflammatory bowel disease, celiac disease, and colorectal cancer, among others.

hello you can use this article

Mahin Homayoun Cibi, D.M., Hausenloy, D.J., Singh, M.K. In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart. J. Vis.

Exp. (110), e53993, doi:10.3791/53993 (2016).

Using two different cell lines in culture studies can offer several advantages and help researchers obtain more comprehensive and reliable results. Here are some reasons why researchers might choose to use multiple cell lines:

In summary, using two or more different cell lines in culture studies helps researchers account for biological diversity, validate findings, and enhance the overall reliability and applicability of their results.

Hi, it is difficult to predict your results and where you have to go. For plants regenereration through organogenesis or somatic embryogenesis you have to be certain according your objectives of your research. Should be to explain better?

Thank yoiu

Thanks a lot for your thoughts, Apostolos. I appreciate it. I guess I'll have to do some post-processing then.

Yes, there are several FcR blocking reagents that can be used in vitro to block Fc receptors on human macrophages. Fc receptors are involved in the binding and uptake of immune complexes and play a role in various immune responses.

One commonly used FcR blocking reagent is human immunoglobulin G (IgG). IgG can saturate the Fc receptors on macrophages, preventing them from binding to other antibodies or immune complexes. Human IgG can be obtained commercially and used at a high concentration in the culture medium.

Another option is the use of monoclonal antibodies specifically targeting Fc receptors. For example, anti-CD16 (FcγRIII) antibodies can block the FcγRIII receptor on macrophages. Similarly, anti-CD32 (FcγRII) antibodies can block the FcγRII receptor. These antibodies can be obtained from commercial sources and used at the appropriate concentrations for blocking.

It's important to note that the choice of FcR blocking reagent may depend on the specific Fc receptors involved and the experimental setup.

All the best with your experiment

The kit and worked well but depending on the cell lysis buffer.

PBS with 1% Triton X100 was fine, but with 1% SDS absolutely destroyed the signal.

The final concentrations of detergents in the LDH assay reaction mixes were much less (about 0.03%), so suggest you carefully check that the sample buffers do not interfere with the assay.

The difference between assessing EC50 in the presence of physiological levels of human serum albumin and alpha1 acid glycoprotein vs. % human serum is that the former takes into account the effects of specific serum proteins on the activity of the drug, while the latter does not.

When examining the in vitro antiviral activity of a drug in the presence of a series of dilutions of human serum up to 40 percent, the % human serum approach only considers the overall effect of the serum on the drug activity, without distinguishing between the effects of different serum proteins. This approach can provide a general idea of how the drug behaves in the presence of serum, but it may not reflect the actual physiological conditions in which the drug will be used.

On the other hand, determining the EC50 values in the presence of physiological concentrations of α-acidic glycoprotein and human serum albumin provides more specific information about how these serum proteins may affect the activity of the drug. Both α-acidic glycoprotein and human serum albumin can bind to drugs and affect their pharmacokinetics and pharmacodynamics. Therefore, by examining the EC50 values in the presence of these specific serum proteins, it may be possible to gain a better understanding of how the drug will behave in vivo, and to optimize its dosing and administration for maximum efficacy.

Plant preservative mixture (PPM) solution is commonly used for sterilizing plant tissue explants in tissue culture.

It is generally not recommended to reuse the PPM sterilization solution because it can lead to reduced effectiveness in sterilization over time. This is because the active ingredients in the PPM solution may degrade or become depleted with repeated use, leading to a lower concentration of the preservatives that are necessary for effective sterilization.

It is recommended to use freshly prepared PPM solution for each sterilization, and to avoid storing it for extended periods of time. The PPM solution can be stored at 4°C for a short period of time (a few days to a week), but it is best to prepare fresh solution as needed.

If you need to sterilize a large number of explants, you may need to prepare multiple batches of PPM solution or increase the volume of PPM solution used for each sterilization.

In summary, it is not recommended to reuse PPM sterilization solution, and it is best to prepare fresh solution for each sterilization to ensure maximum effectiveness.

Another possibility is that the compound was not soluble at the higher concentration and precipitated/crystallized.

Hi! I don't know why, but I cannot see the answer that somebody gave you and now I've got the same question you had haha, is it possible if you could send me the information about how to dissolve cerulenin??

Thank you very much

You could try using different source tissues for the micropropagation. Seedlings may have little latex present and would be a good source of fresh growing cells. Seeds are also relatively easy to surface sterilize and grow aseptically.

If at lower concentrations it had no effect, I always add a solvent control at par, in this case you could use sterile water if your extract is aqueous and do the viability test and the result would show that the extract is the one with the activity.

As you are trying to sterilise(surface) plants growing in water, maybe try in a smaller "pond" to grow them in water added chlorine

Solution for sterilisation. And also try to have shoots growing above the water level.

As per the INVAM website and literature chicory root will be the best whoever the technology has been tested using roots of many crops like carrot, tomato....

Thanks for the answer Max Winkeljohn , Alberto Tosca I'll try your recommendation.

You can use the method suggested by Silvia Di Santi. You should change the media every 24hr. If the parasitemia is high, split some and keep it below 5%, we usually keep it around 2-3% unless we want to perform any antimalarial assay. Most importantly, you should check the parasitemia level using microscopy every 2-3 days intervals at least and provide fresh RBCs while splitting. For higher parasitemia, you can use inactivated serum or plasma from AB+ donors.

Below is our article for a brief explanation and synchronization method as well:

Dear Doaa Yamani,

you could try to do immunoprecipitation experiments with, e.g., anti-phospho-Y or anti-phospho-S antibodies, to pull down all phosphorylated proteins in your samples. Then do western blotting and use your antibodies for the nonphosphorylated proteins to detect any enrichment after compound treatment of your cells.

There are several commercial kits available, but in principle it comes down to incubate your cell lysate with the first antibody that will bind to all phopsphorylated proteins in your sample (PY-20 detects phosphorylated tyrosine). Then you capture that complex with, e.g., Immunoglobulin binding Protein A or G coupled to either beads or agarose. After washing the pellet, resuspend your sample in sample buffer for western blotting, the bound proteins are released and loaded on the SDS gel. Detection is done with your antibodies for the nonphosphorylated proteins you mentioned in your question.

You have to be careful though during the detection step, since the Immunoglobulins (the heavy and light chains) used for the pull-down experiment can cause problems during the second detection step (check the species your antibodies are derived from to minimize cross-reactivity!).

Good luck with your experiments,

Christian

Hello Fa Ro

You may please refer to the research article attached below. It will be helpful.

Best.

In general, all things associated with tissue culture need to be properly sterilized. For me, I autoclave the complete media (MS, hormones(I use 2.4-D, NAA, BAP, Kinetin), and agar) along with the culture vessel (petri dish or test tube). But it is better to filter sterilize (.2 micron) the hormones and vitamins (of the media) and add them to MS media (agar mixed) when the temperature drops to about 50 degrees celcius.

Dear Maduka Wehella have you read the paper from Bhojwani (1980)? Inside you can find many key tips. Consider genotypic factor: it's more important than nutritive formulation; hormones and doses, at least in my experience. Besides other reports, also check E. R. Joachim Keller and Angelika Senula Micropropagation and Cryopreservation of Garlic (Allium sativum L.). Be patience, garlic seems easy but it's not. Good luck!!!!!

Dear @Reza Ghahremani

Different plant species, such as C. canephora, A. thaliana, and Musa spp. responded successfully to the Somatic Embryogenesis induction using different explants, conditions, and concentrations of PGR. The details can be accessed at:

DMEM typically contains a pH indicator (phenol red)- the media change its color depending on pH - yellow below pH 6.8 to bright pink above pH 8.2. You could check ph in your media. Above are some discussions. Good luck.

Dear Mônica Bossardi Coelho

The StarPac bags are sealed and held for 2 weeks in a growth room at 25 °C with a 16 h photoperiod provided by fluorescent lights (40 μM m-2·s-1). Then the StarPac bags are transferred at 4°C in low light. You can work with 12 to 16 h of photoperiod.

Best Regards,

Jean

Please what did you later do to get the cells to grow? Please how do I also get P.falciparum culture? I want to test the effect of my research drug on the growth of plasmodium cells in vitro

Take a look at this video link

Dear Georgios Formozis

The cytogenetic and genetic stability of the regenerated plants is one of the key prerequisites for efficient clonal propagation. Plants regenerated from relatively undifferentiated callus cultures possess a vast array of genetic changes. Such variations can result in useful agricultural and horticultural products. But sometimes, the variations in traits other than those of interest may be undesirable. Anyhow after regenerating plantlets, you can go for genetic fidelity assessment to understand the level of genetic variation.

Thank you Alwin Prem Anand A

miRNA of interest is not expressed in the recipient cells and I saw a robust overexpression with miRNA mimic transfection (qPCR) in these cells. ISH sounds like a pertinent method to try. Thanks again!

Dear Reza Ghahremani

Minimal plant tissue offers many advantages over big explants. In case of TCL, cells are directly in contact with the nutrients, and have maximum chances of organogenesis and somatic embryogenesis on optimized growth regulators.

Yes, the nature of the color and its intensity can affect somatic embryogenesis experiments and may result in many differences among the obtained explants.

Actually, silver ion is a blocker of ethylene receptors, competing for the same subtract, so I guess that in these in vitro studies the accumulation inhibitory effect is indirect, given the autocatalytic nature of ethylene.

Meristem cells in shoot apex are devoid of pathogens like bacteria, fungi or viruses.

Suggest meristem tip culture to obtain pathogen free plants you are working with.

For details of this procedure read plant tissue culture book.

Sir,

To the best knowledge of me, this company has a wide range of chemicals, some of them can be seen in perfect articles in some well-known journals, such as Cell.The products from ChemFaces, China is OK, but you have to make sure that you are contacting a qualified and honest dealer or the correct contact information of the manufacturer.

Good luck!

Tomás María Tessi Thank you for your input!

That is exactly what we wanted to do initially however we need to do a treatment to plants that should be 24-48h and I do not think the plants will survive it. Or they would be so stressed that the whole experiment will be biased.

So I am thinking about alternative methods. But perhaps I can do the treatment and transfer them to the liquid MS+biotin as biotinylation itself is 2-6h.

Thank you and good luck in your research too, maybe I just needed to look at it from different angle :)

Just be sure to use sterile water for any rinses, no point in doing all the hard work to try and get surface sterile plants just to add back contaminants from non-sterile water.

Here is another possibility. It might not be contamination with an organism or virus. When you disperse your cells with trypsin to subculture there may be some small clumps of cells passed to the new flask. Incomplete dispersal. The cells in the clumps die but do not affect the growth of the other cells. I would have to see the actual cultures to be sure, but make sure that your trypsin/EDTA (or whatever you use) is new.

Dear @Amina Ilyas You may find a detailed discussion related to your query at the link given below as to why 70% ethyl alcohol is used for surface sterilization:

I would like to add that I am fully convinced with the explanation by @Yuan-Yeu Yau on that thread.

Hello, Amina!

We use 70% ethanol for 1-2 minutes, then treatment by 70% bleach for 15-20 minutes on a shaker and further rinsing in sterile water 1-1,5 l (running through sterile sieve). This procedure always was good for seeds sterilisation even cotton seeds. Good luck.

Dear Georg!

Please You look at the article and protocol:

Culture of rat brain capillary endothelial cells This method has been adapted from previously described techniques (Roux et al. 1989; Szabo´ et al. 1997; Deli et al. 2000). Briefly, 2-week-old Wistar rat cortices were dissected free of meninges and minced. All animals were treated according to protocols evaluated and approved by the ethical committee of INSERM, France. The dissection was performed on ice and in phosphate-buffered saline (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4,8mM; KH2PO4, 1.5 mM). The cortices were cut into very small pieces (1 mm3 ), homogenized with a 5-mL pipette and digested in a mixture of collagenase/dispase (270 U collagenase/mL, 0.1% dispase) and DNAse (10 U/mL) in DMEM for 1.5 h at 37C. The cell pellet was separated by centrifugation in 20% bovine serum albumin/DMEM (1000 g, 15 min) and incubated in the collagenase/dispase mixture for 1 h at 37C. The capillary fragments were retained on a 10-lm nylon filter, removed from the filter with endothelial cell basal medium supplemented with 20% bovine plasma-derived serum and antibiotics (penicillin, 100 U/mL; streptomycin, 100 lg/mL) and seeded on 60-mm Petri dishes coated with collagen type IV (5 lg/cm2 ). Either 4 lg/mL puromycin was added for 2 days or 3 lg/mL puromycin for 3 days. Puromycin was then removed from the culture medium and replaced by fibroblast growth factor (2 ng/mL) and hydrocortisone (500 ng/mL).

Of course, the more cells are obtained from a younger animal, the more sensitive they are to antibiotics.

You subculture in a liquid medium and keep on a rocking surface so that the clamps will separate to give you individual cells. You need to find the LD 50 and the GR 50 in order to establish the optimal dose for your callus by exposing to varying doses or different exposure times.

You may need to do regular subculturing. Or you may use non infected plant parts of your in vitro grown plants to start fresh culture. Using antibiotics may affect the culture. You may also think of starting the culture through callus induction. You may later check the genetic homogeneity of the plants using molecular markers.

Dear Begüm Kurt ,

Thank you for your contributions to my question. It has developed my point of view. (like always) I wish good look to you too, it was nice to be your colleague :)

Dear Angelia!

Please You can see in these articles:

Not only the light intensity but also the the quality/source of light influence the growing shoot..for example:BLUE light strongly inhibited the elongation. Incandescent light promoted both growth of stem diameter and elongation of leaf petioles (From:Reference+my own experience)

Hello Sonia, very good question. But I don't have idea in this field.

We cover the seeds of various crops with metal nanoparticles (trace elements) for better germination, development and increase yield. https://www.researchgate.net/publication/281065009_Stimulation_of_Seed_Viability_by_Means_of_Dispersed_Solutions_of_Copper_and_Silver_Nanoparticles

You may have a look at the literature below for nanostructured lipid carriers with vitamin D

For vitamin D alone incubated in Caco-2 cells, I recommend 10 nM 1, 25D3 for 24 hours as a physiological condition, or you can try 100 nM 1, 25D3 for 24 hours to check the maximal changes.

I worked quite a lot with rh and rm proteins in vitro. The answers depend on the source of the protein if you are purchasing the recombinant protein make sure that it is carrier-free or at least that the carrier not harmful to the cells. Many cheap rh proteins sold as control proteins (i.e. ELISA, WB) are usually not useful for cell culture application due to preservatives such as sodium azide. Then as suggested by Simon Caulton you need to determine an effective and non-toxic concentration of your protein to the target cells. Another important question is how the cells are supposed to interact with the target protein. In the case of the cell membrane proteins with the known receptors is fairly easy to get meaningful effects by simply adding the protein to the cell culture media. For many proteins that are located within the cytoplasm or nuclei, however, as well as proteins that are subject of activation or posttranslational modifications this approach is usually not effective and overexpression by plasmids is preferable. The direct delivery of the protein to the target cells by lipofectamine is possible, but usually not effective due to the harmful effects of the transfection process to the cells that usually mask the effects of the target protein.

Dear Ricardo,

Please see these papers:

https://www.researchgate.net/publication/9086099_Lignin_Biosynthesis

https://www.researchgate.net/publication/236143776_Lignin_Biosynthesis_Studies_in_Plant_Tissue_Cultures

https://academic.oup.com/aob/article-abstract/74/5/471/2587355?redirectedFrom=fulltext

Best regards,

Jean

Interesting question, carvacrol is the subject of my doctoral thesis

Michel Branchard you can scan the two papers into electronic files first. Then you upload the files into ResearchGate.

Hello, Many methods exist. Cancer cells have unique mechanical properties that can be tested all-mechanical or optomechanical. Also, cancer cells can also be identified visually by shape or by optical scattering.

If you have the possibility to measure cell hydration is also a way for differentiation. The cancer cell always has higher hydration rate than normal cells

Goog luck!

No, you should use transmission electron microscopy, assuming the particles are electron dense enough to be seen.

Add kanamycin (10 μg/ml) to medium for 10 days and transfer them to fresh medium without antibiotics.

We are working to propagate some citrus rootstock in vitro. We noticed that subculturing every 4 weeks is better in growing plants because the medium of cultivation during this period is depleted. However, the period of subculture depeded on the plant species and culture conditions.

Mouse islets are commonly used in diabetes-related studies. Adequate amounts of good quality islets are prerequisites for a reliable investigation. Three major manipulations are employed in the islet isolation procedure: in situ pancreas perfusion with collagenase, pancreas digestion and islet purification. The whole procedure takes 30-45 min for each individual mouse. By using this protocol, a reasonable number of islets can be obtained in a relatively short period of time. This protocol has been proven to be practicable and reproducible. It can be easily followed by individuals who do not have previous experience in the related research field.

thank you Alyona M. Alexandrova I will try that.

Kindly go through this useful PDF attachment.

thanks for replying dear Wassan m.hussen

Required amount of Nanoparticles you can simply add to your culture media, or you can make stock solutions of Nanoparticles in sterile water and dilute according to your need to add in your culture media. In each case ensure that your nanoparticles are throughly dispersed. Ultrasinication is good option for mixing properly. In some cases Nanoparticles unique characters may get degraded due to heat, so preferably donot autoclave the nanoparticle solution. Add it after autoclaving the MS media. Nanoparticle solutions can be filter sterilized separately.

Well dear Mohamad Issaoui , of course the need is to clone any of these plant species. However, in the starting question, there is no information as to WHY germinate seeds under in vitro conditions. To do what? They don’t germinate at all under in vivo conditions? Species rescue?

However, if ever it is to clone them, the worst thing that one can do is to germinate them. And that’s where the difference between Angiosperms and Gymnosperms come in the discussion. Any Angiosperm sp can do something after germination, what is not the case for Gymnosperms. And ontogeny has nothing to do in the story. It is a question of physiological age. Yes, physiology is the expression of genes through biochemistry, cytology, morphology, etc. Nothing new! Except for geneticists!

What is the point?

Dear Parth Desai .....

you can decrease the Light intensity for a week in first sub culture, or also use low concentration of GA3, that will effective in elongation of your new shoots

Best regards

Please look at the following below links which may help you in your analysis:

Thanks

5-Fluorouracil

Retinoic acid

inositol hexaphosphate

Correct...

Hi,

As an undergrad I used to make calli from tree buds coming from outside and I know contamination can be a huge problem. We used the following method: 1min. 96% ethanol, 5min. HgCl (not sure if it is allowed everywhere although it is not volatile or anything and as long as you don't ingest it , it is fine), 3x5min sterile water. I think for seeds ethanol can be left even longer. As you already know that you have a lot of fungal contamination you could also treat with a fungicide prior to the actual sterilisation.

If in your conditions neurons strongly attach to the substrate, the method that you use for harvesting neurons will cause cell damage. I think that it is likely that you have early and late necrotic cells, because late necrotic cells show a more damaged cytoplasmic membrane and incorporate higher amounts of 7-AAD compared to early necrotic cells. Also, it could be cellular aggregates. To be sure, first discriminate aggregates (FSC-A vs FSC-H) and then you can make a FSC -A vs 7-AAD dot-plot . Late necrotic cells will be smaller and with higher fluorescence for 7-AAD, respect to viable 7-AAD negative cells. By contrast, if what you are obtaining is a cell cycle profile of the dead cells, as Waqas suggests, you will find that cells with a higher fluorescence for 7-AAD tend to be bigger. In any case, FSC -A vs 7-AAD dot-plot will allow a better discrimination of live and necrotic cells because you will analyze two parameters instead of one.

Dear Samsher,

Unfortunately (or luckily), ROS are complicated and a collective name of many different reactive molecules. I don't work on mammalian systems but plants. Similarly, ROS bursts can occur upon entering hypoxia, but may decline (depending on the type of ROS) upon reaching anoxia depending on the type of ROS and organelle/tissue studied.

I would always suggest looking at their methods and check how and when they did their measurements and draw careful conclusions from there.

Best of luck

thank you.

What do you mean "in vitro culture" in pot or cal in Petri dishes ?

Abstracts:

1- Effect of diluted sea water on growth, yield, essential oil productivity and chemical composition of coriander (Coriandrum sativum L.) plants.

2- Efficiency of chemical, biological fertilizers and gibberellin on Coriandrum sativum L. under the condition of salinity and calcareous soil.

There could be many reasons for lack of in vitro CTL tumor killing:

HLA or H-2 mismatch

Leukemia cells may lack TAP

Down-regulation of MHC on tumor cells (check with labelled Ab)

CTL lack perforin or Granzyme B expression (check by Flow Cytometry)

I know there are metabolic pathway shifts in fungi when they are grown under those two conditions. The fungi may be inhibiting uptake of the inhibitor, inactivating the inhibitor or may just be insensitive to it under growing conditions as a submerged culture.

Hi again, if the exogenously added TB4 is somewhat different from the endogenous (tagged, labeled with a fluorescent probe) you might still consider localization experiment.

Dear Rakesh,

I want to collect the pituitary gland of catfish in normal laboratory condition after fully acclimatized for the in vitro experiment.