- 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
- Alejandro Díaz-Moscoso added an answer:Spouse working in the same lab or environment
Let me put earlier question in this way: Earlier question was Should researcher marry another researcher;
My alternate question is that would you prefer or not prefer your spouse working in your lab or in your close vicinity?
There is a very good historical example: Pierre and Marie Curie worked together towards the nobel prize (and also their daughter did the same with her husband) and they are recognised as a really in-love couple (even after his accidentally death).
In my personal case, me and my wife (recently married) have been working in as a couple for 10 years, and everything is OK. We started in the same lab and have managed to move together (always working in the same city) through e different cities in 2 countries. We never needed to "do deals" with anyone, each of us have managed until now to get our own places and funding. It may need some concessions, but like any other relationship and, if the relationship is well based, you will find the way to succeed in your careers to the best of your possibilities and will be no jealousy for the other achievements, but proud.Following
- Mohd mohd mustafa Mustafa added an answer:Collagen type I coating issue - can anyone help?
I have been trying to coat my chamber slide wells with collagen type I produced by sigma aldrich cat.no:9791 but I have some problem with it. Under the microscope I saw degraded structure of collagen. I was able to see proper appearance of it once before.
I store collagen which is dissolved in acetic acid at -80°C. After dissolving into ice, I dilute collagen solution with 1xPBS. I coat wells, incubate them for 3 hours in flow, remove supernatant and let wells dry. Unfortunately, wells are not coated successfully. May someone suggest a solution to me please? Thank you in advance.
There are many procedures that can be used to coat Transwell inserts with collagen or other biological coatings. The following is a simple protocol designed to produce a thin coating for cell attachment and cell spreading. Refer to your collagen supplier’s protocol for additional collagen coating procedures.
◗ Type I Collagen Solution from Rat Tail (Sigma Cat. No. C3867)
◗ Phosphate buffered saline (PBS)
◗ Sterile diluting solution (ddH20, PBS, 70% Ethanol, or 0.02M Acetic Acid)
◗ Corning Transwell inserts
◗ Pipettors and sterile tips
Note: Due to the viscosity of most collagen we recommend making a diluted stock solution first and then using this solution to make a working solution at the final desired concentration.
If the stock is made at a high concentration such as 2 mg/mL, it can be stored at -20ºC for
long term storage or 4ºC for short term storage.
Note: Different cell lines will require different collagen coating densities in order to obtain the desired results. We recommend optimizing the collagen concentration and coating time for your cell line and experimental needs. In our studies we found that coating density as well as coating time had a significant impact on cell spreading.
1. Dilute collagen solution to desired concentration with diluting solution.
Example: to coat a 96 well HTS Transwell plate at 10 µg/cm2 from a 2 mg/mL working solution Convert desired coating density (collagen/cm2) to collagen concentration (collagen/mL): 0.143 cm2 x 10 µg/cm2 = 0.0572 µg/uL 25 µL
Calculate total collagen needed to make 3mL of working solution: 3 mL x 0.0572 mg/mL = 0.1716 mg of collagen
Calculate volume of stock solution needed:
0.1716 mg = 0.086 mL of 2 mg/mL stock solution 2 mg/mL
Add 2.91 mL of diluting solution for a total of 3 mL.2. Add the appropriate amount of diluted collagen solution to the Transwell® insert
(see table below):
Table 1. Recommended Coating and Washing Volumes Insert Surface Recommended Recommended
Transwell Area (cm2) Coating Volume (mL) Wash Volume (mL)
96 well HTS 0.143 0.025 0.05
24 well 0.33 0.05 0.1
12 well 1.12 0.25 0.4
6 well 4.67 0.6 1
75mm insert 44 5 8
3. Allow inserts to dry in biological safety cabinet, partially covered.
4. Aspirate any remaining collagen solution from the inserts and rinse once with PBS or cell
culture medium (See Table 1). The inserts are now ready for use or can be stored at 4ºC.Following
- Peter V. Zolotukhin asked a question:Custom organic synthesis services
Dear colleagues, could you please suggest trustworthy custom organic synthesis enterprises according to your experience?Following
- Lee Siggens added an answer:Have you ever tried to transfect THP-1 cells? How high is the transfection efficiency?I have tried to transfect THP-1 cells (human monocytic cell line) with a plasmid containing the GFP reporter gene but I could not get a high transfection efficiency. I also tried electroporation (Neon Transfection System) and the transfection efficiency was higher. Although the cell viability was lower, almost all of the cells alive were transfected. I analyzed only by fluorescence microscopy but I could analyze by FACS.
I would like to know if there is someone used to work with THP-1 cells and if I could get some tips about how to work better with them.
Hi. THP-1 cells are notoriously difficult to transfect. Without using virus based methods electroporation is probably best. Invitrogen's neon system is really nice but initially expensive. It works well with other blood and leukemia derived cell lines as well as hard to transfect primary cell lines. Invitrogen also claim to have some success with lipid based reagents - see here - http://www.lifetechnologies.com/se/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html - good luck.Following
- Tamer Ali added an answer:Does anyone know a good efficient protocol to isolate nuclei from Drosophila larvae nuclei to be used later in native MNase-ChIP as well as 3C assay?
thank you Ivette, Cell culture protocols can not be applied on larvae .. i checked that myself...do you have any other ideasFollowing
- Jose Gros added an answer:Aneuploidy in cancer "stem cells"?I have a question concerning aneuploidy in cancer "stem cells".
In most tumors one could observe cells unevenly divide their genetic material and producing aneuploid cells but in most "normal" cells aneuploidy is lethal. I know that there are some mutations allowing cancer cells to survive mild changes in chromosome composition but I believe that aneuploidy that goes too far must be lethal to cancer cells as well. So there should be some kind of balance - mutations increasing fitness but not to much. (so I believe most cases of hugely uneven genetic material composition that we can observe in microscopic analysis of tumors is only a by-product and these cells die just after division).
But how it is in case of cancer "stem cells"? Are they similarly aneuploidic as other "somatic" cancer cells or keep their chromosomes more under control?
You may like this:
- Simon Knoeb added an answer:Can anyone help with setting up a "no reverse transcriptase" control for qPCR?Are these 2 scenarios the same?
Setting up a "No RT" control in a 2-step qPCR is done by:
1. including all components in the reaction mixture but omitting the reverse transcriptase enzyme when doing a cDNA synthesis in the 1st step.
2. using RNA in the place of cDNA when doing the second step of the qPCR.
I just wondered about the usefulness of this -RT control. As Ubanako suggests, one can simply use the isolated RNA to check for cDNA contamination. The only difference is, that you do not treat the samples in the same manner, when you want to compare them with +RT cDNA samples. But the gDNA content should be the same, right? So if the sole purpose is to check for gDNA contamination, taking the RNA should work similarlyFollowing
- Victor Calvo-Perez added an answer:Co-authorship or authorship to technicians in Research manuscripts
Scientists have their own rules, there is no defined rule which technical person gets authorship? What do you follow or is being followed in your research vicinity or lab?
I recently asked a "dude" down here to proofread a paper I need to send. He included himself as an author. Regardless of the quality, shouldn't that be reported within the "Author Contributions" ACS style
"The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.‡These authors contributed equally. (match statement to author names with a symbol)"
I don't think proofreading is a "work" needed to be included. Where do you draw the line?Following
- Anne Hermans added an answer:Does puromycin enter yeast (cerevisiae) and what concentrations has anyone used?We want to try the ribopuromycylation method for locating active translation in cells e.g. David et al., 2011. JBC. 286. 20688-20700
We are also interested in using puromycin for quantitating protein translation in yeast. Is S.cerevisiae sensitive to puromycin or is a PDR5 knock-out strain required?Following
- Tim Tolentino added an answer:What is single cell biology
I came across a question from my superior "what is single cell biology". I tried answering him saying that studying the biology of cells that are obtained from a single cell and he is not happy with that answer. Can any body help me
I like to keep things simple, so my answer is going to be simple. You were asked, "What is single cell biology?"
Well, since Biology, at its core, is 'The study of life' or 'The study of living things' or 'The study of living systems' etc....I would say that Single Cell Biology is 'The study of the life of a single cell' or 'The study of the systems within a single cell and how they maintain the life of a single cell.'
I noticed that the question did not say, "What is the biology of A PARTICULAR cell?" or "What is the biology of this very specific cell?"
I love studying biology at the cellular level. To me, each and every cell is a very real LIVING thing. Whether it is a single-celled organism, or a single cell of a very complex animal. Whether we look at a bacteria or an epithelial cell from a human being or a lung cell from a cheetah or whatever, the ALL are living systems; They ALL:
- reproduce in some way- use energy- have complex structures- respond to environmental stimuli / interact with their surroundings
So, if the question of, "What is single cell biology?" were addressed to me, my first response would be, "The study of the systems that maintain and make the life of a single cell." Every cell is a living thing that on some level eats, sleeps, breathes, metabolizes, reproduces, and basically tries to keep doing so as long as it can...
Good luck to you.Following
- Valeria Avdoshina added an answer:Does someone know if there are human dopaminergic or cholinergic cell lines, if so, where are they available?
MN9D and SH-SY5H cells are dopaminergic ( with or without differentiation)Following
- Satyajeet Salunkhe asked a question:How cell recognize that all origine of replication fired .
it was one of the question asked in classFollowing
- Vahab Nazeri added an answer:Does freeze drying change enzymatic or non enzymatic activity of antioxidants in plant tissues?I want to freeze drying plant samples and after that measure the activity of antioxidants in plant tissue. Is it maybe?
Thank you very muchFollowing
- Liaque mohammed Latif added an answer:Why did my HEK293 growth rate reduced after transfection?
This maybe nothing, but after transfection my HEK293 growth rate reduced. Has anyone experienced this, and would this be of concern? I used Roche Extreme gene HP transfection reagent and 1.5ug of my plasmid at a 1:2 ratio, in a Six well cell culture plate.
Thanks for all your advice. Just let you all know, appear managed to get things working. In the end all it required was a 700ug/ml G418 dose to kill all of the control cells and allow the resistant cells to flourish. All transfected cell line are now growing fine.
- Debamitra Chakravorty added an answer:Is there any software for predicting disulphide bonds in a protein?I want to see the possible disulphide bond formations in a particular protein.
Disulfide by Design
DiANNA : http://clavius.bc.edu/~clotelab/DiANNA/
GDAP: a web tool for genome-wide protein disulfide bond prediction: http://www.doe-mbi.ucla.edu/Services/GDAP.Following
- Anita E Livio added an answer:When can I store the solution in a protocol I used? step 4 or step 5?
PROTOCOL (For Cells)
1. Preparation of cells.
Note : After preparation of adherent cell or suspension cell in 50ml tube, centrifuge at 2,000- 3,000rpm for 5 min. Then wash cells with PBS/DPBS (optional). After washing, count cells and use approximately 5x 106 cells.
2. Harvest the cell pellet by centrifuge at 13,000 rpm for 10 -20 seconds.
Note : After centrifugation, remove the remnant using a pipette.
3. Resuspend cells in 400 ㎕ PRO-PREP TM solution, and mix well.
Note : Depending on tissue types, one can vary volume of PRO-PREP TM solution. Generally add 5x106 cell per 400㎕, but determine the optimal amount of solution according to cell size. Also, pipette carefu lly as the addition of PRO-PREP TM solution can produce bubbles.
4. Induce cell lysis by incubation for 10 -20 min on ice or freezer at –20℃.
Note : PRO-PREP TM solution don’t freeze at -20℃, and it can be stabilize protein refraining protein degradation with protease inhibitor. Before incubating, it can also increase cell lysis using a syringe(opti onal). At this time, there appears bubbles, yet doesn ’ t need to care because they disappear during centrifuging or incubation.
5. Centrifuge at 13,000rpm (4℃) for 5minutes, and transfer supernatant to a fresh 1.5ml tube.
6. Measure of protein concentration.
Note : When measuring protein concentration by Bradford’ method etc., PRO-PREPTM solution is made to have no absorbable hindrance, and so can decline an absorbable error.
Which step can I store the solution in a protocol I used?step 4 or step 5?
Either after step 2 before you add the Pro-prep and then thaw on ice to continue or, as people have mentioned, after step 5 when when you have collected your lysate.Following
- Vadreenath Tripathy added an answer:Can anyone help with the cytoskeletal dynamism of a cell?
Suppose a cell oscillates between two different shapes(morphology) then how to study what all changes are going on?
thanx everyone for ur beautiful suggestions, I'll try to go through some literatures and hope to get the holy grail...thanxFollowing
- Massimo Bonora added an answer:Staining calcium and endoplasmic reticulum in cancer cellsWe would like to stain both calcium and endoplasmic reticulum in cancer cells growing in the same plate (dish or flask) and by using only one protocol so that we can merge images to locate calcium (inside ER or in cytoplasm). Is this possible? Can you recommend products-protocols for this imaging process?
are you interested in staing Ca2+ into the ER or into the cytoplasm? They are two extremely different pools.Following
- Saquib Ashraf added an answer:How do you prepare and use ascorbic acid in cell treatment?As we all know ascorbic acid is very unstable in aqueous solution because of its oxidizing behavior, can anyone please tell me if I want to do treatment with ascorbic acid to cell lines then how could i prepare it? Can I prepare it in water and can I store it in +4 or -20/-80 for further use? Do you treat it every after 24 hours or 48 hours?
Prepare 2-5 ml of stock solution don't store it because it will be degrade, use as per your requirement and discard the rest.
Try to add ascorbic acid in the last just before the incubation to avoid the light.Following
- Eleonora Vannini added an answer:Blockade of DSCR1?Does anyone know if an antagonist exists for DSCR1?
- Jean-Francois Têtu added an answer:How do I get rid of fibroblast from endothelial cell culture?
I use primary HUVEC for my experiments. After immortalization of these huvecs, i notice that fibroblasts are outpopulating my HUVEC. Does someone know a toxin that i can use to selectively kill fibroblasts while leaving the huvec alive in a cell culture?
Hi, some enzymatic solutions are also available on the market, under the Fibrout brand name. This is easy to use.Following
- Jakub Novak added an answer:Can anyone suggest how to bring the rest calcium level lower?
I am using HeLa cells loaded with Fura 2-Am, and my measurement buffer is Kreb Ringer one, containing 1.3 mM CaCl2.
Which concentrations of BAPTA-AM are you usually using (and in which cell type)? What is the experimental setup - does it have to be i.e. preincubated overnight or something like this? Thank you!Following
- Pendyala Chitralekha added an answer:How can I increase germination in Arabidopsis embryo lines?
I have some Arabidopsis line with less than a 10 % germination rate. I have tried to increase the germination extending the stratification period (until 2 weeks) and treating with GA. The ratio didn´t increase. I´m now wondering if my seeds have normal embryos or not but I cannot find an easy way to see the embryos (I have mature seeds already). Could anybody help me with this? Maybe an staining?
Thank you very much!
If you want to see the embryo inside the seeds, you can use the technique of clearing and staining method given by Stelly et.al., 1984, Stain technology Vol. 59: 155-161. or by Young et.al., 1979, Can. J. Bot. vol. 57: 1668-1672. Worked with tomato seeds.Following
- Sachin Kumar added an answer:Can anyone recommend me a ELISA TGF-beta 1 kit to test human serum samples?
I would like to quantify the systemic levels of TGF-B1 in human patients. My previous experience was with Biolegend in primary human cells but I'm not finding clinical papers that used the kit from Biolegend. R&D systems's kit has much more citations. Has anyone any experience on cytokines measurement on human samples?
I used R & D ELISA Kit and recommend it to you too.Following
- Palanisamy Rangadurai added an answer:Can anyone recommend the staining protocol for Canine oocyte penetration test?I am working in Canine frozen sperm-oocyte penetration assay for last 2 months. Still, I could not get better result for aceto-orcein staining method.. Can anyone recommend the staining protocol for Canine oocyte penetration test?
Dear Prof. Travnik,
Thanks for your valuable suggestion. Hope, this will improve my research work. Which stain is more suitable for the zona penetration test? Can you give me the protocol for the same..
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