- Natascha Fussi added an answer:What is the best method to detect aggrephagy and mitophagy in a human cell line?
Detection of mitophagy and aggrephagy in mammalian cell lines.
Hi Ru-Jeng Teng,
do you know when the new guidelines for detection of autophgay are coming out??Following
- Susana Rodrigues added an answer:How can I successfully culture NR8383 rat alveolar macrophage cell line for adherence?
I am trying to utilize NR8383 cells, and need them to grow in attachment. The NR8383 cell line is partially suspended and partially attached when it is grown. It has been suggested to culture only the adherant cells, however it seems there are far less adherent cells.
Does anyone know how to successfully grow adherent NR8383 cells? I appreciate any thoughts and ideas.
I have seen different medias used, and I wonder if this could play a role in their attachment. We use Ham's F12K supp with 2 mM L-glutamine, 15% heat inactivated FBS, and 1% pen-strep.
Is there any literature about the optimization of these cells?
Thank you for your help.
yes I have 4 t-flasks growing and planning to freeze 2 of it tomorrow and check viability and only latter on decide what to do with the others, for other cell lines I have already follow that protocol of keep some cells "alive" until check if stocks are fine. I think I will follow your advice and in 1 flask freeze only adherent cells and for the other both and check which one work better.
Once again thank you TrevorFollowing
- Reza Yarani added an answer:Does anyone know if 4T1 cell line (Mice Breast Tumor Cells) have Her2/neu expression or not?4T1 cells can simulate the stage 4 of human breast tumor in murine model. I wonder if anybody can help me to know if this cell line has Her2/neu expression on their surface membrane or not? Also I'm about to find that this cell line has estrogen dependency in in vivo model? Does it look like MCF7 cells (estrogen dependent)?
4T1 cells are Her2-negative. To get Her2-positive cells you can buy special 4T1.2eRB2 cells from AntiCancer Inc, (http://www.anticancer.com/Fluorescent_protein_cell_lines_April_2010.pdf)Following
- Mary Hamilton added an answer:Can anybody suggest an immunoprecipitation protocol using Dynabeads (invitrogen)?I have to do mass spectrometry after protein complex elution.
I have no answer. In fact, I need advice! I have not used this technique, but have acquired Dynabeads to try.
MY problem is the need to identify a protein for which I have an antibody, with the possibility that my unknown protein is NOT the antigen, but a different protein in what is a fraction separated only by size on Sepharose CL-6B. Electron microscopy is the only method for its detection. If I immunoprecipitate the known antigen, can I find the unknown in what did not precipitate???Following
- Yolanda Calle added an answer:Sites for exchange of skills or access to equipment.Does anybody know if there is something similar to this but for UK based labs?
Thank you Simona, I will have a proper look in the site.
- Lale Evsen added an answer:How reliable is NeuN for labeling neurons? Is it possible it labels other cells too?I am not sure if the possibility was ever exhausted that it labels other cells like glia.Following
- Dominique Liger added an answer:Enzyme cleave off sulfation on cell surface, is sulfation important or not?
I am looking for any enzyme that has the potential to cleave off sulfate group on protein or cell surface. I am studying alternative glycan structures of pancreatic cancer and asking the question: whether sulfation is important or not?
if you type sulfatase in your favorite search engine, you will find diverse available sulfatases but mainly described active on carbohydrates but I guess it would be worth trying their potential activity on proteins...Following
- Massar Alsamraae added an answer:How long can EHEB and NALM-6 cell lines survive when frozen in liquid nitrogen?
How long do cells stay alive without defrosting them?
you can put your cells in liquid nitrogen for ever, but when you want thawing them try to keep them away from stressFollowing
- Muresanu Cristian added an answer:How many mitochondria can be found in a healthy human sperm cell ?Human cells may house anywhere from 2 to 2,500 mitochondria, depending on tissue type, antioxidant status, and other factors.
Very good paper. Thank you Marika.Following
- Menno ter Huurne added an answer:Can I get high quality RNA from PFA-fixed cells?
I want to fix my cells with PFA, store them at 4°C, then sort them and isolate RNA for sequencing. Is this possible?
Thanks for the answers / advise,
I did the sorting (indeed with the FACS), and ended up with 0.25 - 4 *10^6 cells per sample. As a control I also took along fresh (unfixed) cells. I added trizol to all samples and put them in the -80 freezer. Tomorrow I will first isolate the RNA from a fixed and a control sample and I'll see how it works out. I'll let you know.
- Ketil winther Pedersen added an answer:Major aggregation of Dynabead M-280 Tosylactivated prior to coupling antibody?
Our lab has received several orders of Dynabeads M-280 Tosylactivated that have major aggregation prior to coupling the antibody. Has anyone else been having these issues?
I am working as a staff scientist at the production site for the Dynabeads.
Aggregation of beads can happen but is usually more of a ”cosmetic” problem as the actual application is not affected.
In general, short sonication is a good way to reduce aggregation of the beads and insure optimal homogenous conditions at the time of ligand addition when coating the beads. When target is bound to the beads one needs to be more careful as the binding might break.
The surface charge of the beads may in some samples give problems with "floating" or "sticky/aggregated" beads. The stickiness may be due to electrostatic interactions between the beads or the beads/tube wall. This makes it quite hard to work with the beads - they become difficult to handle. Aggregation/stickiness of the beads we usually recommend to wash the beads in a non-ionic detergent like for instance Tween 20 before doing the experiment. The problem is usually reduced/removed by simply adding non-ionic detergent like e.g. Tween 20 to a final concentration of up to 0.1% to the beads, followed by resuspension and washing in buffer without Tween 20. Incubation in the Tween 20 solution may be needed, e.g. 5-10 minutes in room temperature on a roller. In addition we recommend using normal siliconized tubes. This treatment will most likely reduce the electrostatic potential of the beads and hence reduce the aggregation/precipitation.Additionally, try to keep pH as high as possible for your experiment and salt concentration low.
Beads left on the surface of the sample (floating on top) may be due to surface tensions which stops the beads from moving to the magnet - however in these cases only a very small fraction of the beads are left on the surface and the rest of the beads have been moving to the magnet.
In some cases it seems that the aggregation starts when adding the Abs. One might consider to use the indirect method and pre-label the cells with antibody. This should solve an aggregation problem caused by the addition of the antibody to the beads
Very small amount of beads may remain on the surface of the supernatant and do not respond to the magnet. This phenomena is most likely due to electrostatic charges between the tube and the beads and can overcome by using either siliconized eppendorf tubes or by simply adding non-ionic detergent like Tween 20 to final concentration of 0.01 -0.1% or little higher to the beads followed by resuspension and incubation on roller for 5-10 minutes at RT.
Feel free to contact me at any time at email@example.com
- Aneesh Chandran added an answer:Can anyone suggest a protocol for phagosome isolation?
I like to isolate the phagosomes of MTB-infected macrophages for my study.There were so many protocols available but people who have practical knowledge may able to suggest a best protocol.
Thank you all for your valuable suggestions.Following
- Chad Robinson added an answer:How do you stain plasma membrane domains with Alexa Fluor-conjugated Cholera Toxin Subunit B?
Has anyone tried to visualize the plasma membrane with Alexa Fluor-conjugated Cholera Toxin Subunit B using confocal microscopy?
What concentration is best suited for this, and what fixation procedure is most appropriate?
I have used on neutrophils, the internalization was inhibited by staining at 4 degrees C for 15 minutes,wash, then 1% paraformaldehyde 10min, wash, cytospin; then flourmount at least 24hours before imaging. It may be relevant to remember GM1(CTX target) is a lipid raft marker. published Shock 28 (3), 334-338Following
- Mozhdeh Sojoodi added an answer:Does anyone have or know where I can get MIN6 cells in the US?
I would like to use min6 cells for some experiments involving glucose stimulated insulin secretion, but have been unable to get them. Cells are for academic use, and we are willing to either purchase them if they are sold commercially, or pay Fedex shipping if any lab has a vial to share. Thanks.
I have them but we are in Brussels, Europe. Let me know if you have no other choice.Following
- Habibu Usman Abdu added an answer:What would be the fate of a cell with a very low level of the reduced glutathione?
I have just measured the reduced and oxidised forms of glutathione in a mosquito cell. The level of the reduced glutathione was very low just about 30% of the total glutathione. Considering the importance of the reduced glutathione in protecting the cell against oxidative injury, what would be the fate of such cells with very low levels of reduced glutathione?
Thank you all for your contributions. I found them useful.Following
- Tushar Tomar added an answer:Upto which passage number LNCaP can be used for molecular and epigenetic studies?
I want to use LNCaP for some molecular studies.So,I just wanted to know the highest passage number of this cell line which can be used for these kind of studies
Go for low passage number as low as possible let say upto 15-20. As it has been shown by Peter Jones group that longterm culturing already induced quite some chages in the cancer cell lines. But people still uses these cell lines upto 100 passage number and keep publishing the results. However, don't forget to perform STR profiling for authenticity of cell line before you start experiment and within 6 months after fishing experiments. Since most of cancer research journals request that before final submission of article.Following
- Alka Madaan added an answer:Is it possible to culture RAW264.7 and THP 1 cells without 10% serum ?
Is it possible to culture RAW264.7 and THP 1 cells without 10% serum ? If that can for how long do they live? Where can I find answers for these kind of questions? Can any one suggest a book or a web? or give me an answer?
Zina, We have cultured RAW264.7 in 10% FBS in culture flasks. However for cytokine release assays, we have first cultured them for 24h in 10%FBS in culture plates. Subsequently, we have incubated these cells even in 0% FBS for 24 h to bring down cytokine levels to basal and then stimulate with inflammatory agent such as LPS/ConA to see enhanced secretion of cytokines. Cells look absolutely happy and with good morphology!
And yes, after serum-starvation, we have treated cells with LPS+anti-inflammatory agent for a duration of maximum of 48 h, so the total duration without FBS is 3 days; 72 h. Cells look perfectly fine!Following
- Karolina Dwi Setyowati added an answer:How can I grow human pancreatic cancer cells?Can anyone suggest me how to grow the human pancreatic cancer cells like Capan-1, Capan-2 CTPAC and MOH RPMI cell line. I am using DMEM with 15%FCS and still it is very difficult to grow them. Does anyone have any suggestions for growing them?
Are you still in NUS? I would like to ask a favour from you. Is there anyway I can pm you?Following
- Lorenzo Morè added an answer:Can anyone suggest how to prevent cracking on Golgi stained slices?
Hi, after just a few weeks my Golgi stained slices seem to have shrunk and show wide cracks.
Brains were embedded in 5% agarose and sliced 100microm thick with a vibratome.
I used FD rapid Golgi staining and mounted them with Eukitt.
Thank-you Miguel, very helpful.
- Tatyana Prigozhina added an answer:Can anyone tell me how I can successfully culture chicken embryonic stem cells?I am trying to produce chicken embryonic stem cell lines, but after one week they are producing vacuoles, lipids, and finally they are lost. Is it really possible to produce CEs cell line?
Take in consideration that avian cells grow better at 400 C than at 370 CFollowing
- Mona Ellaithi added an answer:What are the signaling pathways that n-myc is involved in?
I would like to know if n-myc pathways have been fully or partially identified.
Look at those two papers, I hope they are of use to you
- Eric van der Graaff added an answer:How can I prepare a suspension of urease (3.5 kU/L) ?
I have a solid urease, how can I prepare a suspension of this enzyme containing 3.5 kU/L?
The ammonium sulfate ensures stability of the enzyme upon storage at 4C. In general, repeated freezing and thawing of enzyme in water severely reduces enzyme activity. The final concentration of the ammonium sulfate should be 3.2 M ammonium sulfate at pH 6.0. In practice, we could not come above 2.9 M, so dissolved the (lyophilised) powder in very small volume of water and added this 2.9M stock. The enzyme will precipitate, forming a suspension that appears milky, so you should mix well before pipetting. When diluting this for your assay mix, the protein will dissolve properly.
- Luis Felipe Buso Bortolotto added an answer:Is LDH assay able to evaluate possible mechanism of action related to necrosis?
Since LDH assay determines membrane integrity, may this test be sufficient to conclude that there is a necrosis cell death-related induced by drugs?
These are interesting points. Thank you all!Following
- Nicole Kühl added an answer:Can anyone help me with cell isolation and culturing?
I have some experience in cell isolation and culturing them. However those are mostly short assays (1-2h) and no real stuff. I want to know what culture medium is best for macrophages, lymphocytes, hepatocytes, cardiomyocytes, glial cells...so normal cells no cancer cells! Also can someone suggest methods for isolation of hepatocytes, cardiomyocytes, glial cells...What enzyme to use for tissue digestion? Some people use colagenase type I while other use IV?
Next thing is what to do with FCS - some people do heat inactivation while others don't...so what to do?
Are the any other tips that can be useful! I have read many papers and talked to a lot of people that have experience in cell cultivation but I'm still puzzled...
If you are about to start, there are a million points to consider and most of them are cell-type-dependend.
As a start, I would vote for a book like the one from Ian Freshney see at:
He also has published books on primary cell isolation. So if your cell-types are in them, this might be another source. Besides that, each isolation is so special, that I would always try to find a lab where to learn. Otherwise it might take ages. I spent 6 months on primary oligodendrocytes until my cell numbers were high enough.
For many primary cells, special media are much better than DMEM or F12-based one. However, they are also much more expensive.Following
- Alessandro Poli added an answer:What are the good markers for G2/M phase of cell cycle?I was getting high G2/M population in PI staining. I was looking for a specific marker (FC/IF/WB) of G2/M transition. Cyclin B1 and CDK1 usually acts in this stage. Can anyone please suggest a marker?
I am new on research gate, sorry if the conversation is out of date. However, as marker of the G2/M phase you can use some phospho-Cyclin B1 or phospho-cdk1/cdc2 antibodies. You can also try to separate nuclei from cytoplasms, Cyclin B1 accumulation in the nucleus is very high at G2/M, in particular the phosphorylated form (you can see it with IC as well). As control, you can also check the expression of Cyclins D, whose levels decrease at G2/M checkpoint.Following
- Shubham Goel added an answer:Can cells in trypsinized condition(single cell suspension) communicate with each other like cells in culture?
The cells have the ability of to communicate with each other in culture. This communication can be contact dependent and independent(secreted factors). Is anyone aware if cell-cell comunication can also occur under trypsinized/ cell susupension condition? If yes what kind of communications are more prominent?
I have also the same query. i agree with the answer of corriden. pls let me know does trypsinization affect on cytokine receptor ? as i am also looking at the signaling by looking at the phosphorylation status just after the trypsinization of the cell line but its not phsphorylating that molecule even after stimulating with its specific stimulator.Following
- Himadri Singh added an answer:Does anyone know a webpage that lists biomarker by disease?
This may also help:
- Kathryn R Ayscough added an answer:Are there actin-independent endocytosis pathways in fungi?There are some examples of actin-independent endocytic pathways in mammalian cells. What about fungi? Are there any studies on fungi?
I am not aware of fully actin independent pathways in yeast or fungi. The reason for actin begin required is thought to be connected to the fact that their plasma membranes are under tension due to turgor pressure (see Aghamohammadzadeh & Ayscough, 2009). When mammalian plasma membranes are under pressure, endocytosis also now appears to require actin (Boulant 2011). There might be pathways in yeast that have a different extent of requirement for actin (see Prosser et al, 2011 and Aghamohammadzadeh et al, 2014) but complete disruption of F-actin appears to stop endocytosis. Currently however we know very little of these alternative pathways and their requirements for other proteins such as clathrin for example.Following
- Prasanta K Ray added an answer:Our lab needs to perform couple of experiments using the LAPC-4 cell line. Would anyone be willing to kindly provide them to us?
LAPC-4 cell line.
Please contact the cell banks and commercial suppliers.Following
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