Tissue Culture - Science method

Can shoot & root production be done directly from leaves without callus stage when initiated in tissue culture media with appropriate hormone. If so what hormone combination can be used?

Hello all,

I am using Sytox green dye for dead cell counting using a Live cell imaging instrument. As this is a non-permeable dye for live cells, I expect to get a very low count in untreated cells (Background). When I treat the cells with different cell death inducers, it should give high counts eventually. But many times, when I add Sytox green to the wells containing untreated cells, immediately after that, I get a very high count. Sometimes the count goes down after few hours, sometimes it stays the same. I am using around 100 nM Sytox green concentration. Cells are macrophages. Could you please help me regarding this if anyone has encountered such problems and have any solutions. I am using 12 well tissue culture plates, around 1 million cells per well.

Thanks and regards,

Prabuddha

I'm working on Mentha longifolia (horse mint) and trying to transform it using Agrobacterium via tissue culture. However, I'm having trouble growing it from seeds on MS media. The seeds either don't grow well or they become contaminated. DDoes anyone have experience with horse mint and can provide the whole method for successful tissue culture?

I am trying to detach CFBE cells from tissue culture. I either use RIPA or cold PBS to scrab cells but I couldn't get the enough pelete

1. Should I use coating media because cells are not attached properly and removed by washing

2. Can I collect the media and don't discard it

Please give me any remedy

All plants are green but some of these plants becomes yellow. I did not found any reason. Please help me to find out the real problem.

To combat environmental contaminants, plant tissue culture labs are fumigated. Would vapourised hydrogen peoxide have a negative effect on the tissue cultured plants in jars?

I will be assisting my colleague to grow MCF7 cells. 75cc flasks need to ordered first.

Will this flask work for MCF7 cells ?

This flask is tissue culture treated.

I saw another flask that is cell bind treated (which is more expensive). So have the confusion if the cheaper tissue treated flask would suffice.

This question might sound silly, but the right flask needs to be ordered and work started asap !

Thank you in advance for your replies!

I have been trying to grow Nicotiana benthamiana via tissue culture. I used ethanol and sodium hypochlorite with different concentrations and time duration for seeds sterilization and lastly washed the seeds with distilled water but there is no growth.

At some concentrations there is no contamination but the seeds didn't respond.

Currently, I have only Ethanol and sodium hypochlorite as sterilizing agents.

Could anyone help me to grow N.benthamiana using these agents?

Thanks

Regards

Nadeem Ahmad

Im working on oil palm tissue culture we are using MS media along with IBA and NAA hormones for platlets but the response was not that good can anyone suggest if any changes needed in media and hormone combinations that we can try to improve root growth in oil palm?

Strain: LBA4404

Growing media: YEB (pH 7)

Temperature: 28°C, 200 rpm (overnight culture)

Antibiotic use: Kanamycin and Streptomycin

Everytime I centrifuge my H6c7 cells, the cells don't pellet properly. Though there is a pellet formed, the supernatant has visible particles which I believe to be the cells. I usually centrifuge at 300 x g for 5 mins. I have tried increasing the centrifugation time to 10mins and also increasing the speed to 500 x g. Despite this, I always lose cells dramatically. Please has anyone had a similar problem with this cell line, or another cell line? If so what method did you try to solve the problem?

Hi Everyone!

I am currently designing primers to validate my CRISPR-KO mammalian cell line using TIDE. The primer is designed about 250bp upstream of the PAM site. Then, Sanger sequencing is run using the primer on DNA extracted from the KO cell line. The TIDE software then deconvolutes the indels in the mixed KO population to tell you the efficiency of the CRISPR on the gene of interest.

TIDE software: https://tide.nki.nl/

In order to make the primer sequence specific to a region of genomic DNA (an intron) upstream of my PAM site (in an exon), I think that I should make the primer sequence longer. What is the max length you would make a Sanger primer to obtain specificity? I am using BLASTn to check the primer sequence against the human genome. Even once I have a specific primer, how can I validate that it does not have off-target binding sites in the human genome? Is there an experimental validation that should be run first? Or, will the purity of my Sanger read upstream of the cut site show me that I have only sequenced the target? What are other recommendations for TIDE sequencing primers?

Thanks!

Hello! I want to measure the osmotic pressure/osmolarity of some tissue culture media. I have a freezing point osmometer which I use for liquid media, however I don't know if this will be appropriate for tissue culture media as it contains agarose (0.8% w/v). I have considered measured the media before addition of agarose, but I am not sure if the agarose would change the outcome. Does anyone know how to handle this?

Thanks :D

I am working with SHSY-5Y and using NEST-made tissue culture plates. I am looking for some companies that may support stronger adherence to these poorly adherent cell type.

Can we collect scions from 1-year-old grafted plants instead of collecting scions from 40-year-old trees (candidate mother plant)? Are both plants physiologically and genetically the same? If yes, can we go for invitro shoot tip grafting (STG) by using scions from the grafted plants?

I would like to add Fungin (Invivogen) to my MS medium to control fungal contamination. Unfortunately, I cannot find any documentation for the product that says whether to add it before or after autoclaving the medium. I don't imagine it is heat stable, but I wanted to double check before I go through the hassle of adding it to individual culture tubes. If it is not heat stable, how cool should the medium be before I add it? Can I add it to cooled medium immediately before culturing explants?

Thank you!

Dear all,

I want to express my protein of intterest in absence of salt. Now the challenge is to see the survival of cells and even if it survives, where I could find a cell medium (any company that provide it). Please recommend if any commerically available media that has no salt.

Thank you

With kind regards

Prem

I want to know which part of the bamboo shoot will be taken after the root

HELP!

Our lab has recently discovered these dancing blobs in our TC cultures - across cell lines, primary cells and organoids. So far they are not behaving like any bacteria/fungi/yeast we have ever come across (not responding to antibiotics/antifungals and no turbid media). They seem to be amorphous and both extra/intracellular..

Someone has suggested they may be protozoa? If anyone has seen something similar or is an expert microbiologist please help us identify them!!

(Picture included for attention but really need to watch the videos to distinguish from cells/debri)

Hello,

I'm working on plant tissue culture. Sometimes the tissue I cultivate produces what you can see in the photos attached.

I'm wondering if these are also calluses, or they can be organ regeneration?

Thanks in advance for your precious comments

Ok so I've been maintaining Eucalyptus species callus cultures, and have run into issues with phenolics, so I've tried to make media (MS) with 1g/L activated charcoal, but it seems that the AC has reduced the gelling capability of the medium substantially. The medium is semi-solid (I used 4g/L agar to keep the medium a little squishy but I didn't want it THAT squishy

any tips? Maybe add a little KOH to balance the pH and improve the agar's gelling capacity?

I am working with a nitrogen sensitive species trying to induce somatic embryogenesis. The textbooks I've gone through so far insist that casein hydrolysate - or another reduced nitrogen source - is crucial to initiating embryogenic callus. but many of the protocols I've read don't include CHL or any other nitrogen sources. So what's the truth here? Is this reagent crucial or not? I've ordered the casein hydrolysate just in case, but am trying to figure out if I'll even need it.

please with protocole/article

Our Antibody Tissue Culture department has had issues with Myco contamination in the past. We are currently having to dissassemble our Clarifying and Concentrator TFF units to soak the Millipore filters and concentrators every weekend. The idea is to avoid risk of myco contamination of filters/concentrators.

However, disassembling the units this often is an ergonomic concern.

My question is: Is there a better way to handle this issue?

My suggestion to the team is that we allow the cleaning buffer to run through the filter in reverse and forward motion for a longer period of time, as opposed to letting them soak over the course of two days. I consider the motion to be efficient in cleaning the unit thoroughly.

Any suggestions?

Hello,

I am using the Sartorius Vivaflow 50R cassettes for tangential flow filtration. They are reusable, but it doesn't say how many times they can be reused. We are running batches of 500mLs of tissue culture medium containing B27 (no FBS). We've been able to reuse them 8 time so far without much drop in the time it takes to concentrate our conditioned medium.

What are other people doing? Thanks!

Hello all,

We cultured MSCs on calcium phosphate discs for 3 days and 7 days. We are seeing strange crystal-like precipitates or something of the sort (images attached). They are found wherever cells are found, or nearby cells, that are growing on the surface of the discs. We did EDS on these samples out of curiosity and the crystals appear to have a high concentration of NaCl, which indicates that they are salts.

I can't find any literature that shows this happening in their cell studies. Has anyone else seen this sort of thing happen in their cell cultures? I have no idea what could explain these results and I would appreciate some insights, or hypotheses, if any.

Thanks!

Since I opened a new frozen stock I am getting a lot of contamination. I have checked everything- medium, PBS, trypsin etc. Everything looks good.

Dear researchgate members,

I recently made two attempts to grow the aquarium plant Cryptocoryne wendtii emersed, i.e., outside of water. Unfortunately, both attempts failed, and I am unsure of what went wrong.

In the first attempt, I heated soil in the oven and shaped it into a cube. I then placed the aquarium plant into this cube. In the second attempt, I used rock wool instead. In both cases, I lightly moistened the soil and rock wool with aquarium water. Subsequently, I placed them in plastic bags and provided CO2 by exhaling into the bags through a straw. The bags were sealed with rubber bands and positioned under an LED strip light. The distance between the light and the plants was approximately 10 cm, ensuring that the light intensity was not harmful.

After one week, I exchanged the air inside the bags and provided more CO2 by breathing into them again. Unfortunately, after two weeks, I couldn't observe any positive results. Almost all the plants in both the soil and rock wool died. There was no growth observed, neither in the plants themselves nor in the roots.

I am very confused and frustrated, as I don't understand where the mistake lies. Do you have any ideas or advice on what I might have done wrong? Are there specific conditions that I should consider to achieve successful emersed cultivation of Cryptocoryne wendtii?

I would greatly appreciate your help and support! Thank you!

I did today tissue culture for mcf7 cell line( breast cancer) and i check my work under microscope i saw this below is this cancer cell or just cells debris?

Our colleagues are preparing for their 3D spheroid culture and they plan to use 1~1.5% agarose solution to coat the plate. When choosing the agarose for use, there are different kinds to agarose with different melting point, gelling points and EEO properties. There is no guidance about which type of agarose is suitable for plate coating. For those with a gelling point at 36 ℃, we have some concerns about melting of agarose coating at 37 ℃ in the cell culture incubator chamber. For those who have experience on this field, which type of agarose should we choose?

Plant propagation refers to the process of reproducing plants to create new individuals. In plant breeding, several methods are employed, including sexual and asexual propagation techniques. Sexual propagation involves the use of seeds or spores to produce new plants, while asexual propagation involves vegetative methods such as grafting, cutting, layering, or tissue culture. Each method has its advantages and is chosen based on the specific breeding objectives and characteristics of the plant species.

I am planning to do an Mn2+ (500uM) treatment for my HeLa cells. However, when I tried to prepare Manganese(II) chloride tetrahydrate stock (dissolve MnCl2 · 4H2O into PBS), I found that the stock in PBS was cloudy and my treatment seemed did not work properly.

How can I make the stock properly in PBS?

Hello everyone,

I am sorry in advance for my english, it is not my mother tongue.

I am a student who's currently working on a protoplast regeneration project. Unfortunately there's a problem with the medium I'm using.

I am trying to develop a protocol for protoplast regeneration from a plant (can't say which one). I've started since 4 weeks and last week was the first time of trying a new medium.

I used the next supplements:

- MS medium with Modified vitamins (100%-50%)

- 1% Sucrose

- 9% Mannitol

And after autoclaving this I added:

- 10mg/L Zeatin

- 10mg/L 2,4D- or 50mg/L NAA

I used different concentrations of MS medium with modified vitamins.

I had 9 different media:

MS 100% with standard sugar (as stated above) (with zeatin & NAA) (did not crystalize, but had crystals in medium)

MS 75% with standard sugar (as stated above) (with zeatin & NAA) (crystalized)

MS 50% with standard sugar (as stated above) (with zeatin & NAA) (crystalized)

MS 100% with standard sugar (as stated above) (with zeatin & 2,4D-) (did not crystalize, but had crystals in medium)

MS 75% with standard sugar (as stated above) (with zeatin & 2,4D-) (crystalized)

MS 50% with standard sugar (as stated above) (with zeatin & 2,4D-) (crystalized)

MS 100% with standard sugar (as stated above) (with zeatin & 2,4D- and active charcoal (0,02%)) (did not crystalize, also no crystals in medium)

MS 75% with standard sugar (as stated above) (with zeatin & 2,4D- and active charcoal (0,02%)) (did not crystalize, also no crystals in medium)

MS 50% with standard sugar (as stated above) (with zeatin & 2,4D- and active charcoal (0,02%)) (did not crystalize, also no crystals in medium)

I used 300 ul protoplast suspension with 2,5ml medium.

I will try to use 5 ml next time but I don't know where the crystalisation is coming from. Does anyone have an idea?

I am facing the severe bacterial contamination after 2 to 15 days of sub-culturing of sugarcane as this is noticed as yellowing of leaf in attached photo. Can anyone help me how can I save these plants. Photo is attached below

I am doing 3D tissue culture. These black dots show up in my spheroids from time to time, please take a view of the picture below. Does anybody know about these black dots?

What is the frequency or rate of somaclonal variation in terms of percentage or per 1000 plants in tissue cultured plants?

Can anyone guide me about oil palm tissue culture protocol using male inflorescence as explant? Explant sterilization, Suitable media and its composition, Hormonal combinations?

How is the use of LED lamps in tissue culture economically?

#light #tissueculture #micropropagation #growthroom

Is there anyone working on oil palm tissue culture?

How canI know if this media is good?

I am dealing with human epithelial cancer lines cultured in T-75 flasks. After trypsinisation, trypsin inactivation and centrifugation, the cell pellets collected were resuspended 30-40 times with fresh media before subculture/seeding. Nevertheless, clumps of cells can be observed along with single cells.

Kindly advice to avoid the cell clumps.

1) Any better angles for the tubes containing pellet and pipette during resuspension?

We wish to set up a tissue culture lab but we are constrained by space available to us as a dept (Plant Science & Biotechnology)-We are trying to squeeze in all the lab units into the available laboratory space; tissue culture , molecular biology and plant pathology labs. We have designed the proposed lab with all the units in place taking all the safety precautions into consideration in the design. There is no issue with tissue culture and molecular biology labs all in place under the same roof but my worry is about including plant pathology lab even though it can be accommodated because of the problem of contamination. As the head of department i want to carry everybody along; the plant science ( for plant pathology) and the biotechnology ( for plant tissue culture+molecular biology) staff for logistic reason and otherwise. With a background in biotechnology this is the dilemma I am facing trying to squeeze in all the three lab units against my worry about contamination. Pls i need advise and suggestions from experts in the field of Plant tissue culture , molecular biology etc on this proposed multi-purpose lab. Attached is a sketch of the lab.

Two important things about this method confuse me.

1. some of the researcher say ascorbic acid and citric acid are heat sensitive, so cannot be add to MS medium before autoclave.

2. 2nd thing is pH, of medium, if i add these antioxidant after autoclave in laminar flow unit, how can i manage the pH of medium, because it will change the pH of the medium.

I am using a specific brand of MS Basal medium with vitamins and I see that my plants are not growing as fast as they should. I am attaching the medium composition of the brand I am using and the one I would like to try. Any recommendations?

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!!

For spraying 0.1% bavestin on plant let's of sugarcane, tissue culture.

Checking of sugarcane production and productivity by tissue culture method, I want to know the answer. As I read and study, this techniques couldn't create genomic modifications. Please, share your views?

You are carrying out virus infections. Your stock contains

5 x1010 pfu/ml. What volume of this stock would you use

to infect a tissue culture flask containing 2 x107 cells at

an multiplicity of infection of 5 pfu/cell?

Tissue culture treated or non-tissue culture treated microplate.

Thanks.

Hi, I have problem to sterilize freshwater plants (Anubias, Bucephalandra). I used chlorine salts and ethanol. I got contaminations. I think AgNO3 could works. Do you have any other protocols for sterilization?

Thanks for all responses.

Bohuš

I've been having a bit of trouble with yeast contamination for DU145 cells. I purchased nystatin to rid the contam but nystatin will not be able to help with any potential bacterial contam. Is it possible to use both the AA solution and nystatin or just leave out the AA?

The molecular weight of Lamivudine is 229.29 g/mol

Entecavir Mol wt: 277.279g/mol

I am trying to characterize the viscosity of newly developed cell culture media formulation. WE have AR2000Ex Rheometer. I am wondering what could be the values for the parameters to setup for the characterization, especially (a) Range of shear rate (1/s); (b) Gap between the plates; (c) Normal force; (d) diameter of parallel plates; (e) angular velocity (w); and any additional parameters I should consider? Really appreciate any inputs which can help us to get started.

Thanks,

Bhushan

Hi Everyone,

I am culturing the monocytes derived from the PBMC of the patients blood samples. After two weeks of the culture growth, some of the cells appeared as shaded (or in background looks like covered by a layer of something else), [pic 1- day 1, pic 2- day 6, pic 3- day 13 and pic 4- day 21] imaging at 0.4x.

Is the cells need passaging? If yes, I am worried weather the these cells will be adhered again or not. But as per the protocol, no need to do the subculture.

What should be kept in mind while fumigating a Tissue Culture Laboratory?

I would like to hear from experts that,

how many grams of KmNO4 are and how many milliliters (ml) of Formaldehyde (formalin) to be used how much area of laboratory space?

e.g if a laboratory dimensions are like L=50, B=30 & H=12

for this laboratory could you please suggest the require quantities of KmNO4 and formulin

Some where I read that 1:2 (17.5 gr KmNO4:35ml formulin) and ratio of KmNo4 and Formulin works better but I didn't find for how much area?

https://www.emtech-systems.com/technical-talk/the-use-of-formaldehyde-in-fumigation/#:~:text=Adding%2035%20mL%20of%20formalin,potassium%20permanganate%2C%20never%20the%20reverse.

Please help

Hi Everyone,

I have an issue with change of culture media color. I prepared culture media A and B. After few days, color changed to pink (B). Any possible reason for the same.

Hi Everyone,

Should I add L-glutamine during media preparation if the culture media (RPMI 1640 with l-glutamine) already contain the same?

Is there anyone working on sugarcane tissue culture, shoot tip culture ?

Good day,

i have a general question about tissue culture.

I have found the following recipe for Epipremnum Aureum "Marble Queen":

Leaf Explant: MS Medium + 4.54 µM TDZ + 1.07 µM NAA (Thidiazuron in Micropropagation of Aroid Plants by Chen and Wei (2018), p. 105, DOI: 10.1007/978-981-10-8004-3_4)

Specifically, I have the following questions.

1) Do i only need to autoclave the agar with distilled water (I use a pressure cooker for this) and when the agar has cooled down a bit just add the MS, TDZ and NAA and mix it or do i need to autoclave the MS as well?

2) Will the TDZ dissolve in the agar water at all and how hot can the agar water be to add the MS, TDZ and NAA?

3) Is it even necessary to autoclave the water incl. agar (in the pressure cooker) if I clean all the jars with NaClO (sodium hypochlorite)?

Thank you in advance!

I am trying to make tissue culture media by using 0.8% agar powder but median does not solidifying what is reason behind that? How to slove it?

Autoclave temperature set at 121 and 20 min running time

PH set at 5.82

I am attempting to micro-propagate a terrestrial orchid using pseudobulb and leaf segments as explants. Since the callus was induced roughly two months ago, it has not continued to grow. What could be the cause of this and how can the issue be fixed? I would appreciate any and all helpful advice.

Thank you in advance.

I want to use some reagents for culturing immune cells.

I want to be sure these reagents do not have endotoxin/pyrogen contamination.

The purity for a reagent is listed as "molecular biology grade" purity.

Does molecular biology grade indicate the reagent is endotoxin/pyrogen free?

Shall we have previous genetic variation in explant tissues used in in vitro approaches, how can we estimate an adequate ratio for somaclonal variation among regeneration events?

In tissue culture we use several plant tissues and “reprogram” the cell development to obtain somatic embryos, calluses, shoot meristems, whatever.

Considering that the raw material we start with would not have a chance to follow another developmental sketch (E. g. a spongy parenchyma cell) is expected to die as originally planned, a spongy parenchyma cell.

It is clear that some stress triggers, frequent cell divisions, growth regulators and other substances, may increase the mutation ratio differently. We may also consider that there is a tiny probability of this mutation events occur by chance.

My reasoning is, how can we correctly attribute a somaclonal variation ratio being aware of the possibility of mutations that would not jeopardize a cell developmental path already settled in an explant (a chlorenchyma cell for instance) that, was not for the tissue culture detour, would “kick the bucket” as such (a chlorenchyma cell)?

During Tissue culture of Brassicae when explant(hypocotyl) was provided Ms medium containing NAA -0.125 mg/250ml and BAP-0 mg/250ml. The hypocotyl burst and formed shoot, in direct-embryogenesis we expect the formation of somatic embryos from the explant. Please explain is this organogenesis or somatic embryogenesis. Rectify please if I'm wrong in some interpretation. The snap is attached below.

Thanks in advance.

Regards,

Vinay

I am culturing iPSC-derived as well as primary rat neonatal and adult microglia on collagen IV-coated TC ware. I wanted to perform some flow cytometry experiments and tried detaching cells from the surface with Accutase (40 min incubation at 37C, as described in Reich et al https://www.frontiersin.org/articles/10.3389/fimmu.2020.617860/full#h3), however microglia are still stuck to the surface and even quite aggressive tapping of the well plate does not facilitate their detachment.

Has anybody else faced the same issue and could advise on how to detach microglia from the surface?

Thank you in advance!

what is best method of surface sterilization of explant in tissue culture ? and how to minimize fungal contamination in tissue culture ?

Can I use Fungicide for surface sterilization of explant ?

Which fungicide and Quantity ?