Questions related to In Vitro Culture
I did a water-soluble extract from a fruit and I'm going to use this extract to treat cells in vitro.
In order to test the highest dosage in vitro (2 mg/mL), I will need to use a large volume of extract dissolved in the medium for the treatment. What is the maximum volume I can use in vitro (dissolved in culture medium) which will not disturb the cells and cause their death?
Initially, I put 30 ul of extract in a total volume of 150 ul (20% of extract in medium). Is that too much and eventually can cause cell death? Does anyone have that experience?
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.
I'm currently working on anther culture of tomato and most of embryos I've got usually ended up looking glass-like structure, that makes they are so hard to continue their development into normal structures.
I am currently taking care of Plasmodium falciparum in vitro culture and in order for me to keep them alive, I was wondering how and when should I feed them if you have any protocol to advice me. I have access to complete media and RPMI.
Dear Good People
Is it possible to study the effect of a compound on the activity of a certain signaling pathway but you only have antibodies for the total protein/nonphosphorylated form involved in that pathway! or it would be a waste of time and money?
"P.S."... No abs for the phosphorylated forms are available📷
Thanks in advance!
I was wondering whether NAD+ (not nicotinamide riboside or nicotinamide mononucleotide) can enter cells when supplementing the medium of a neuronculture with NAD+
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!
Hi anyone can help me to find methods to increase the multiplication of garlic plants in vitro culture. I am using Ms media, NAA and BA solution atm?
Various plant growth regulators were found to have a positive effect on organogenesis and somatic embryogenesis, but which one is more important?
What you see in the image is dry extruded faba bean which hasn't been exposed to any treatment (heat or chemical) after the extrusion. The DMEM has been added 12% FBS, 2.5% TMP/SMX, 1% Pen-Strep og 1% Fungizone. This is the result after incubation for 24hr at 37°C.
I know that the DMEM changes color after pH-changes, but I don't understand why it would change into two separate phases and what it is in the extruded faba bean that causes it. I spread it out on TSA-plates and it didn't show any growth of the bacteria that was previously there (B. cepacia and R. insidiosa) or any other bacterium, so the sterilization was deemed effective.
I appreciate any answers and theories.
SLOW GROWTH STORAGE for the mid-term conservation of a large number of species, including tropical and temperates. In this case, reducing the in vitro growth through the application low temperatures and less hours of light.
Anyone can help me with this trouble shooting of plasmodium falciparum in vitro culture? I've been tried to culture plasmodium for approximately 2 months but they didn't grow well. Everytime I check thin blood smears of my culture, they appear like this (see the picture I attached) . It seems they couldn't invade or infect the red blood cells and stay outside. Please give me some advice about what I have to do so they can grow well and the ruptured schizont can invade RBCs.
We know that somatic embryo can be used as an artificial seeds, but encapsulation include many kind of material. I ask to researcher to give their experience for us.
In order to measure the LDH level in the experimental groups in Pc12 cells, I tried the lactate dehydrogenase activity assay kit (MAK066-sigma). I used one on the brand's page for the protocol, but the results were meaningless. There are some studies that used the same kit but different protocol. I don't know what to do. Is there a protocol that uses this kit or that you can recommend?
Hello to everyone! I am trying to multiplicate a specific variety of Kiwi with tissue culture. I managed to multiplicate some. Some of them came from callus and some were produced directly from the starting material. What I would like to ask is, do the plants that come from callus differ genetically from the mother plant?
My experiment involves treating recipient cells (at 70% confluency, 24 hours after seeding) with EVs (adding EV suspension dropwise to the culture media) before assessing a specific miRNA expression in the recipient cells after 24 hours (by qPCR). I see no change in miRNA expression in EV treated vs untreated cells.
From a separate set of experiments we know
i) EVs are taken up by these cells (using dye based method)
ii) EVs are enriched with this specific miRNA
Any suggestions would be greatly appreciated!
Many pieces of researches have shown that responding explant percentage, embryo number, plant regeneration and also response rapidity increase in TCL explants compared to larger size explant. Now, I ask all researchers to share their experiences and reasons.
We know that light has a key role on different plant mechanisms, but its effect on the embryogenesis is question. Thanks for expressing the opinions of researchers.
Due to the fact that silver ion is a blocker of ethylene receptors, some studies have reported that the accumulation of ethylene has decreased during in vitro culture due to their application.
How can this be justified?
Fungal and Bac contaminations are common in vitro culture. However, Ppm works alright with some of the contaminated plants . But bleach and ethanol doesn't work at all. Any recommended solutions or media types?
We are planning for wet-lab experiments in India, so we need a few chemicals reagents. The cost of products is comparatively less in ChemFaces company based in Wuhan, China. Can this company be trusted? will it provide high-quality and authentic chemicals. Your suggestion will be of great help!
Website link of ChemFaces
I am waiting for your valuable reply...
Dear colleagues, fellow scientists and students,
I am working on establishing of protocol for BioID in plants. I am aware of already existing protocols such as cell culture, the transient transformation of tobaccos and root culture. For our purposes, we will need to work with seedlings and I was wondering if someone ever tried to perhaps transfer the seedling onto solid MS media enhanced with biotin. Could that work? What would be a good concentration to start with? I'd be grateful for any insights or links to relevant literature backing up the yes-no argument.
Thank you and all the best in your research!
The use of antibiotics, preservatives and similar chemicals is aimed to be avoided in cultures. Since isolations without the use of antibiotics and preventive chemicals is my goal, latent bacterial and sometimes fungal contamination is encountered. Does anybody has experience treating source plants with any preventive chemicals or antibiotics few days or weeks prior to isolation to reduce or eliminate the internal contaminants?
Any recommendations/suggestions will be highly appreciated.
Hi, guys, I am a postgraduate with lots of experience in culture contamination. However, when I was about to be sure I will never be bothered by contamination, this (as is shown in the pic) occurs！ and spoil my almost one-month effort........I dont know what this is and where they may come from, can anyone give me some help？Please！
Thank you guys for your answers! After several days of observation, yellow objects grew more in number, and there are more and more dead cells in the culture. Some objects are floating while others in inserted between cells. From my point of view, they do not seem like cell debris or any other "healthy" things..........
Although it's not a very effective sterilizer alone for plants but still being part of treatments in all researches. i wonder over its widely essential use and what factors decide the time of ethanol treatment?
Since tween 20 is no doubt an excellent surfactant to remove surface tension and provide better exposure of plant surface for sterilization, by removing the bubbles which are more problematic in case of plants having plenty of hairs and trichomes. I wonder if in case of unavailability of tween 20, can a common detergent or dish soap provides the same benefit or there is any other cheaper compound as a replacement?
Hello! I want to isolate RBEC from a rat, I used the protocol - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208856/. However, I used 5-day old rats instead of adult rats. Subsequently, the addition of puromycin (4 μg / ml) killed 100% of the cells. Could this be due to differences in the age of the rats, or is it worth looking for other errors?
Attached is a photo of a mixed culture, no puromycin.
After in Vitro culture of my sweet potato callus, how do I sample or prepare the callus to be subjected to UV-A radiation and also calculate the dosage used.
In media preparation, the plant tissue culture media needs to be adjusted to ± pH 5.8.
Why it is so and what would happen if the pH is lower than 5 or more than 6.5?
I need to examine the in vitro antiviral activity of a drug in the presence of a series of dilutions of
human serum up to 40 percent (e.g., 5 percent, 10 percent, 20 percent, 40 percent).
An EC50 value for 100 percent human serum can be extrapolated from these data and the serum-adjusted EC50 values reported. In addition, I need to determine EC50 values in the presence of physiological concentrations of α-acidic glycoprotein and human serum albumin.
What will be the difference between the data from the first paragraph vs the second?
I am performing Caco2 permeability experiments using transwell inserts and I am using phenol red permeability assay to verify the monolayer integrity.
What is the maximum phenol red permeation allowed in order to state that there is no damage to the cell monolayer?
I had this problem in my in vitro cultures of Cnicus benedictus, which I derived from immature embryos. After several months of vegetatively multiplying the plants to get a lot of clones from each genotype, which I further want to use for polyploidization with oryzalin, I have experienced endogenous bacterial contamination. The senior doctor, who is a specialist in in vitro culture in our department, says that some embryo cells may already have had endogenous bacterial contamination, and now that I've cut the plant tissue while propagating, we've come across the cells, and the contamination has gone to the media. Does that seem likely?
I use 100 microliters per 100 milliliters of PPM (Plant Preservative Mixture) and, I'm recommended to increase this concentration three times. If that doesn't help, I'd have to use the media using antibiotics, but I'd like to avoid that because it would take me at least a week of passage and another week of passage back to classic MS after a week.
What would you recommend that I don't have a similar problem in the future? Alternatively, what are your pointers for handling the current sticky situation?
Many thanks in advance for the suggestions and good day and good health.
3D Cell Culture is newly optimized and promising discovery in cell organization research and other researches that needs evindences in cell culture (in vitro) level. Are 3D Cell Cultures mimic epithelial cells because they are lining up the specific surfaces ? Is it possible to create an cell culture environment as we want in 3D cell cultures ? (e.g. microenvironment that show inflammatory responses as a result of cancer-related inflammation). Is real-time screening of cell response (e.g. after drug exposure) available in 3D cell cultures ?
I'm studying the immune profiles of persistent/ chronic infections in human PBMCs. I'm interested in knowing if there is any published ex vivo or in vitro model that allows me to do repeated viral stimulation to mirror the potential persistent infection in human?
I know there are humanized mouse and organoids, but they don't seem optimal. Thanks a lot.
One of the main issues in better acclimatization of in-vitro woody plants is "stem thickness". How can we get the ticker stems during multiplication stage?
Doing an in vitro study with nanostructured lipid carriers with vitamin D and with just vitamin D alone, and just trying to figure out the optimal incubation time so I don't stop it too late having saturated the cells and wind up seeing no difference.
I would like to observe the effects of a recombinant protein on in vitro cultured cells. For this reason, I need to know a concentration at which recombinant proteins can be used in in vitro culture without killing the cells. I've been looking for information but I couldn't find a proper answer to my question.
Did anyone ever work with recombinant proteins incubated with cells in vitro?
If so, does anyone have any tips about working concentrations, at least a suggested starting work concentration, from which I can start?
Thank you in advance to you all!
Have a nice day
We are testing the anti-inflammatory activity of these compounds on human PBMCS in vitro. Is there any recommended concentrations that are being used? I would be grateful for some references in that respect. Thank you in advance
1.Technologie des Légumes (Y. Tirilly - C. Bourgeois) Tec Doc 1999 - ISSN : 0243-5624 : Génétique et création de nouvelles variétés de légumes (M. Branchard - M. Pitrat) 27-44.
2. Variability in plants regenerated from tissue culture (E. Earle - Y. Demarly) Praeger1982- ISBN : 0-03-059364-6 :In vitro culture of barley: a method to study Rhynchosporium scald disease and select plants resistant to the toxin, rhynchosporoside (M. Branchard) 343-350.
For certain reasons, frozen tissues are only for in situ staining. Is it possible to use frozen tissues for other purposes? Such as isolating the tumor cells from the other cell types present for in vitro culture. I value any feedback and discussion on this.
if we put nanoparticles inside cell culture media, is it possible to measure when the particles uptake the cells? can DLS measure the particle inside the cells?
seed pre-disinfection and tetrazolium test for select viable seeds to seedling in in vitro culture.
I am working on alectoris chukars' semen in in vitro condition. I want to know dilution rate of its semen for evaluation of semen traits such as motility, viability and etc. Note that I use beltsville poultry semen extender (sexton extender) as semen diluent.
thanks in advance
I want to investigate the effect of some nanomaterials on the plants using in vitro culture. So it's important to make solution of those nanomaterials to use in MS media with different concentration. The nanomaterials I want to use are MgFe3O4 and MoO3.
Normally, the success of seed germination through in vitro culture is influenced by several factors such as medium composition, seed origin (plant species), seed maturity...
The success to ensure normal germination and development from in vitro cultured seeds of perennial lignified plants is very limited. Why??
I am working on Papaya micropropagation where I am getting plenty of shoots but they are not increasing in height. I have tried diffuse light, GA3 but did not see improvement. Please suggest a better treatment.
I am working on production of stevioside from Stevia rebaudiana under in vitro culture conditions. We want to check the cytotoxicity of the plant extracts on cell lines, preferably pancreatic cells. Please advise and is there anyone who can provide us with the cell lines in Delhi
I am looking for a drug that can stimulate TGF-beta expression of epithelial cells in vitro culture. I appreciate your suggestions.
I`m quite fresh in the field of culturing CD8 T-cells in vitro and I need an advice. I want to test if a population of CD8 T-cells may give protection against LCMV-Armstrong virus infection in mice. So, we want to isolate the cells from LCMV-immune mice, expand, then transfer into naive hosts and challenge the mice with LCMV. As a measure of protection we want to use viral titer. We rather doubt if the cells are protective.
The point is that antigen specificity of the overall cell population is not known. The cells of interest contain some antiviral effectors, but relatively few and it`s not known whether these effectors play any role in the response.
TCR-transgenic mice are unavailable as far as induction of the marker I`m studying is not possible to achieve.
So, I`d avoid antigen-mediated CD8 T-cell expansion as far as it can promote some cell while depleting the rest from the population of interest. Initially I planned to use IL15-mediated expansion. Preliminary tests with that cells from naive mice were promising for 50 and 100 ng/ml of IL15. Cells grew very fast and within 1 week I was able to get 5-6 millions cells starting from 0.1 million of sorted cells.
I am making a preliminary trial with total memory CD8 T-cells from LCMV-immune mice. The cells grew quite fast on IL15 and expanded more than 20 times within 1 week. They were viable, proliferated in response to LCMV-peptides and secreted IFNg.
I wanted to expand them more to make some additional tests, but after 1 week of culture in vitro the cells stoped growing. Right now 1 week passed, but they hasn`t expanded even a little. They are just alive, neither die, nor proliferate.
Unfortunately, after stimulation with plate-bound aCD3e (1 mcg/ml overnight) that IL15-expanded CD8 T-cells from LCMV-immune mice died very fast. So I`m not sure if I can use this method.
Any ideas how to expand them? Should I try weak aCD3 stimulation with plate-bound antibodies and cytokines? Or I`d rather try some stimulator cells with soluble aCD3? Or some cytokines? IL15/IL15Ra?
Hello all! I am currently facing a big trouble with fungi contamination in in vitro culture of native forest seeds, because they come from field collection with a lot of endogenous contamination (fungi). The disinfestation with alcohol (1min) + 2% hypochlorite (15 min) does not work, they insist to proliferate ... Would pre-treatment with fungicide or some other solution solve this problem? Any suggestion?
I am a second year graduate student and have been doing flow cytometry on embryonic neurons as a part of my experimental procedures. The neurons were cultured in vitro and allowed to form synapses for 10 days after which I used them for flow cytometry. In order to understand the percentage of live and dead cells, I used 7AAD as a marker. Ideally, I would have expected two distant peaks indicating 7AAD positive (dead cells) and 7AAD negative (live cells). However, my graph has three peaks.It looks like a negative peak and two positive peaks. I am however a little confused about this and wanted help to interpret this data.I am attaching the histogram plot in order to get an idea of what it looks like. If anyone has any inputs please do comment. I would really appreciate your help.
I came across the two contradicting views that ROS levels must be low under low oxygen (microaerophillic) or in absence of oxygen (hypoxia).
Other researcher shows that ROS levels are high due to inhibition of ETC. Generation of superoxide anions by NADH dehydrogenase shown to play role.
I wish to knew what happens under physiological conditions in tuberculosis inside granuloma or macrophage where conditions are hypoxic or similar ?
for last two months I try to culture WB-F344 cell which was purchased from JCRB cell bank. As mentioned in the documents I cultured them with MEM including 10% FBS, but after 4-6 passage the cells were died.
any suggestion ?
I am student at FUM University in Iran is working on establishment of in vitro culture of pomegranate plant (Punica granatum) and has problem with Plant Growth Power. The plants produced in the stage of Proliferation are very weak, with thin stems and narrow leaves. How can we enhance Plant Growth Power (pomegranate plant) in vitro culture?
I need information about biostimulators for seed germination, plantlet growth and plant development of following medicinal plants:
1. Nigella sativa,
2. Trigonella foenum,
3. Coriandrum sativum,
4. Rhus coriaria.
Also, I would like to have some papers about development of stress tolerance of these plants both in vitro and in vivo conditions.
I'm trying to optimize the NRU assay on 3T3 cells as low OD540 reading (about 0.1-0.2) were obtained from previously done experiments. I prepared 50ug/ml stock and incubated it at 37C for 1.5 hr, added to cells and incubate for 3 hr.
- I took the neutral red solution that's incubated at 37C from waterbath into the hood prior to washing of cells with PBS. Therefore, I'm thinking if the solution might get a little bit cool down (less than 37C) while I was washing the cells with PBS and that caused precipitations.
I work in cancer immunotherapy particularly in CAR T-cells. I have leukaemia cell lines such as KO-52, ksaumi and THP-1. They express my target antigen at different levels. My CARs are also active, they kill ovarian tumour cell line expressing the same antigen but not leukemia cells. I am wondering what are the possible reasons for tumour cells resisting killing in vitro.
The inhibition of spore germination and growth of Botrytis cinerea upon treatment with toxic molecule occured on solid culture medium (petri dishes) as predicted before. Strikingly, the spores germinated normally in liquid medium that contain also the toxic molecule at the same concentration (flasks).
How can we explain such observation??
(See attached pic.)
I am a first year PhD student and I am working on a protein called Thymosin Beta 4 (TB4) and using it exogenously to treat injured cells in the kidney called podocytes. I am mainly working in cell culture right now and I am treating the podocytes with TB4 by adding it to the media that the cells are cultured in.
One recurring question I keep getting asked is how do I know that the TB4 is entering the cells? I have seen results when TB4 has been added, so I know that it is definitely having some sort of an effect. Does anyone have any idea of how I could prove that the TB4 is entering the podocytes? Any help would be greatly appreciated.
I want to do the in vitro culture of the fish pituitary under different treatment condition. So, can anyone kindly help me the protocol for the pituitary culture.
we have done red cadamba seed germination in vitro by treating with fungicide and double sterilization with 50 % Clorox and denatured alcohol and inoculate it in MS media with hormone but it shows low germination rate even after a month .
please advice me on how to increase the germination rate and reduce the contamination rate
I'm interested in how the secreted protein MMP-2 might be affecting a certain type of cell. I thought about adding the recombinant MMP-2 protein to in vitro cultures of the relevant cell type at different concentrations, but I can't seem to find any literature describing assays that do this so am not sure how best to approach it. Does anyone have experience of using metalloproteinases in this way?
What are the black structures that appear on Botrytis cinerea mycellium after two weeks of in vitro culture??
I tried to observe them under binocular microscope and I think that they are fruiting bodies but I am not sure.
Can anyone cofirm this ??
I am working on wheat mature embryo callus regeneration..Facing problem with regeneration..Plz suggest me suitable regeneration media(India wheat variety)...Please help