- Prabhakar Semwal added an answer:Does anyone know of any colorimetric methods or any cell culture based methods for detection of synaptic plasticity modulation?
Thank you sir.Following
- Neetika Singh added an answer:Can anyone help with histone isolation from Hela cells?I am trying to isolate histones from Hela cells but my preparation is not coming out clean. I am getting other contaminants along with the preparation. Can anyone please come up with suggestion?
Well by saying , its not coming clean i mean lot of basic proteins are coming along. I am isolating the nuclei first and then acid extracting the histones ..I am aiming at getting a clean preparation where i have only 5 bands of histones. I am straight away taking cells of a confluent T75 flask. I am trying to establish cell culture in my laboratory . So as of now I am not getting into the quantification..Following
- Mohammad Ghasemzadeh added an answer:Sertoli Primary Culture (Technical Problems)I am following step by step a published protocol but I am having some problems to isolate Sertoli cells. See attached a picture of Day 1 culture. Could anyone with experience help me to understand if I am doing something wrong?
This is the protocol
Sertoli Cell Culture (Oliveira, Sousa et al. 2009)
1) Transfer testes to cold calcium- and magnesium- free Hanks balanced salt solution (HBSSf) containing 50 U/ml of penicillin and 50 µg/ml of streptomycin sulphate (pH 7.4) immediately after removal.
2) Decapsulate tissue (2 g) and wash twice and cut into small square pieces (2–4 mm) in a sterile Petri dish
3) Suspend minced tissue in HBSSf (25 ml/g of tissue) in a glass-stoppered 100-ml Erlenmeyer and shake vigorously for 1 min to disperse tubules.
4) Leave the tissue to seattle for 5 min on ice, and discard the supernatant.
5) Repeat this procedure twice to mechanically remove red blood cells and free Leydig cells.
6) Digest the resulting pellet in 25 ml of HBSS with collagenase type I (1,000 U; C0130, Sigma) and DNAse (500 U; D4263, Sigma) and continuously shake (100 rpm) at 32ºC for 25– 35 min.
7) Remove the formed aggregate, wash in HBSSf and discard. Add the washing HBSSf to the cellular suspension resulting from the digestion.
8) Wash the resulting suspension twice and leave to settle completely at 4ºC
9) Suspend the resulting pellet in 20 ml HBSSf with 5 mg pancreatin (P3292, Sigma) and DNAse (500 U, D4263) and digest at 32ºC with continuous shaking (100 rpm) for 15–25 min.
10) Discard the new aggregate formed, and add 0.4 ml of fetal bovine serum to the cellular suspension, which has left to rest at 4ºC for 5 min
11) Centrifuge the suspension at 100g for 5 min.
12) Gently suspend the pellet in 30 ml HBSSf. Repeat this procedure twice, and suspend the resulting pellet in 20 ml HBSSf.
13) Pass this suspension through a glass Pasteur pipette in order to loose germ cells from the clusters and then pellet at 200g for 5 min. repeat this procedure twice.
14) Suspend the resulting pellet 10 ml Sertoli culture medium (DMEM-Ham’s F-12 [HF12];
1:1, containing 50 U/ml of penicillin and 50 µg/ml of streptomycin sulfate, 0.5 µg/ml fungizone and 5% heat inactivated fetal bovine serum) and force through a 19G needle in order to disaggregate large Sertoli clusters.
15) For culture of Sertoli cells, the concentration of clusters on the cellular suspension obtained from the procedure described above has to adjusted to 1,000 clusters/ml plated on 25 cm2 culture flasks (Cell; Sarstedt, Leicester, UK) and incubated at 37ºC in an atmosphere of 5% CO2:95% O2. The day of plating was considered day 0 of culture. Cultures were left undisturbed until day 2.
Oliveira, P. F., M. Sousa, et al. (2009). "Membrane Transporters and Cytoplasmatic pH Regulation on Bovine Sertoli Cells." Journal of Membrane Biology 227(1): 49-55.
you are welcome!Following
- Eila Eila added an answer:How do I increase lentiviral infection efficiency in lymphoblastoid cells?I got a very low LV infection frequency (45% at best) when infecting lymphoblasts. The same LV vector gave a very high infection frequency in other cell types (>90%).
Does anyone have any suggestions?
worked with a concentrated virus (Added RFP for immediate feedback). i tried few concentrations & polybren transductionFollowing
- Gunnar Heiko Dirk Poplawski added an answer:Has anybody tried a high titer infection of RGCs with AAV8 injection in vivo above 1xE+14 and had better results?
Most studies report poor tranduction with AAV8 serotype, but thats what we have at the moment.
thanks for your answer. How high was your titer that you injected?
Thank you so much,
- Thomas E Adrian added an answer:In FMRF amide related peptides, amidation is allowed by the terminal glycine. What could be the role of a tryptophane in place of such glycine?As above.
Amidation would not occur. The enzyme is looking for a consensus sequence, while the glycine residue is the donor for the C-terminal amide group.Following
- Lorena Salvatore added an answer:How can I use 2 different antibodies (for two different antigens) from same origin in IFA?Can you please suggest me on how to use 2 different antibodies (for two different antigens) but from same origin in IFA. Both the antibodies are from mice. I tried the staining sequentially for each antibody but I could see that conjugates have reacted to both the primary antibody. Do I need an extra blocking step between the steps?
I agree with all the people that suggest to directly conjugate one of the two antibodies you have. Or (and for me is the better way) to use two antibodies from different species.
- Edelmiro Moman added an answer:How can I measure the drug concentration in mitochondrial matrix?We have designed a novel family of drugs that target an enzyme within the mitochondrial matrix.
A number of those compounds have been shown to inhibit the target enzyme in cell-free assays. However, only one compound is active in cell-based assays.
We attribute this discrepancy to the fact that most compounds cannot freely access the mitochondrial matrix.
Which would me the easiest way to test this hypothesis? In other words, we would like to have a rough idea of the concentration of each compound in the mitochondrial matrix. It does not need to be very precise.
Thanks to all for your awnsers. From them, and from my own literature research, I conclude that they do not exist well stablished protocols or kits that people are using on a routine basis to do that. Which essentially means that we would need to figure out ourselves a methodology and then validate it.Following
- Javad Mirnajafi-Zadeh added an answer:What is a suitable concentration of pentylenetetrazole for making seizure and epilepsy in mouse?I want to simulate seizure and epilepsy in mouse, but I can't find a proper dose of pentylenetetrazole in saline in papers.
The answer of Beate is almost complete. I just want to say that according to the age of animals you may need different dose. Thus, please check the age of animals used in the articles. Usually the PTZ dose needed in young animals is less than older animals. The other thing which is very important is the time of your experiments. If you can not completely control the temperature and humidity of the animal house of your lab, you can find a lot of differences in seizure parameters of animals. Please try to do all of your experiments in warm seasons (Spring and Summer) or cold seaons (Autumn and Winter).Following
- Jai Ghosh added an answer:What is the optimum laser strength one must use during confocal microscopy?I am dealing with a weak dye and a weak laser. I have previously read that the laser strength should not be more than 50%. Is it a rule or just a normal guideline? I cannot see the signal clearly at this strength however, only increasing the laser strength gives me a good signal. I need to compare cells pre and post treatment so any advice with the laser strength, amplifier gain, etc would be great.
There is no such rule of the LASER strength. It all depends on what data you want .Following
- Marc M. Abitbol added an answer:QPCR: reference gene stable under hypoxic conditions?I'm trying to compare mRNA expression levels of my gene of interest in cells at normoxic vs. hypoxic (i.e. 1% O2) conditions. Usually I would use GAPDH as my reference gene but it seems that GAPDH gets upregulated under hypoxic conditions and therefore is not suitable for use as reference gene. Can anyone suggest a reference gene that has been tested to be stably expressed in CRC cell lines under normoxic and hypoxic conditions? I highly appreciate your suggestions. Thank you.
Beta Actin can be used also in combinationFollowing
- Dhurga Devi asked a question:I would like to know more on Bcl 2 activity in unicellular.. care to share anyone?
Bcl 2 familyFollowing
- Mohamed A. A. Mahdy added an answer:Is there a way to use immunohistochemistry to show changes in metabolism of murine muscle tissue following schema (glucose vs lipids etc)?
I am using the hind limb ischemia model in NOD/SCID mice and would like to measure changes in metabolism to the localised tissue both in a control mouse and mouse with recovered hind limb perfusion following hematopoietic progenitor cell transplantation.
If transplantation of progenitor cells induce lipid formation you can stain with Oil Red O or IHC using perilipinFollowing
- Virginia Claudio added an answer:How could I evaluate the number of cells with protocols other than MTT or CCK8?Do anyone have protocols, other than MTT or CCK8, to evaluate the number of cells? Because the mitochondria might be damaged in my experiment. I'm afraid that the dehydrogenase might work abnormally and cause a misinterpretation of the data.
Hi, a bit old conversation but maybe the participants have a good suggestion for me. I find myself in a similar situation. I am checking for different treatements on adherent cells. I use a 96-well plate format MTT assay. I want to determine how cell numbers (proliferation and apoptosis) change at different concentrations of my drug and at 2 time points. Ideally I would like to do this with the same cells. So i was thinking of using an high content screening flourescent microscope to image and count cells just prior to MTT incubation.
Does anyone know which of the above named dyes and stains might work without interfering with the MTT signal? (maximal absorbance at 570nm).
If I could count e.g. apoptotic cells. vs. total cells would that give me information on proliferation?Following
- Priyankar Sanphui added an answer:Can anyone suggest a good protocol for co-immunoprecipitation?I want to study 2 protein interaction.
thank you nishaFollowing
- Bernhard B Singer added an answer:Which is best method for labeling the exosome (PKH, CFSE, or other antibody tagging)?When I labeled the exosome using CFSE, the green fluorescence dye disappeared within a few seconds. Did that occur due to bleaching? Actually, I want to use a super-resolution confocal microscope (STORM or SIM -nikon). In order to avoid bleach-out of fluorescence intensity, which method is recommended? I'm going to use anti-CD9 (exosome-specific marker) as a primary antibody. Then I will use a secondary antibody (-alexa fluoure dye conjugated). Has anyone used an exosome specific marker as a primary antibody in high-resolution confocal microscope to avoid bleach-out of dye?
I agree with Jin-Xin Bei that there is no specific marker for exosomes. All markers like the often used tetraspanins can also be found in other extracellular microvesicles!Following
- Laszlo Nagy added an answer:What is the cellular source(s) of 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2)?Is it known which cell type(s) produce 15d-PGJ2 and in response to which stimuli? I'm not very familiar with PG biology and I find the literature on this specific matter a bit confusing and contradictory. Any pointers will be highly appreciated.
- Colin Reardon added an answer:Anti-CD11c mouse Ab working in paraffin-embedded sections?We have been trying to get by IHC labeling of CD11c positive cells in our lungs and spleen sample from mice.
The best we could get was a weird labeling in red.
We do not label the cells as the labeling is smaller than the nucleus.
It seems that our Ab is being trapped in some particular unknown structures...
So right now, I am not trusting this can of picture to say that I'm seeing CD11c cells.
Here the following info to understand the pics:
it is confocal microscopy: depth of the plan: 0,125 um.
blue is DAPI
green is an FITC-labelled Ab
The 2 pics show you different fields.
Thanks in advance for your help.
What clone/pAb did you use, can you share your protocol?
our experience has been similar to Klaus Ley'sFollowing
- Nadia Halidi added an answer:Can anyone provide a protocol to collect cell lysate from cells seeded on soft substrate for western blotting?No tripsinization please. I want to collect cell lysate from cells seeded on silicon gel for western blotting. I wonder if anyone has a nicely working protocol for this that avoids the tripsinization step.Thank you all for your answers. That was helpful.Following
- Hanna Björkelund added an answer:Do you have suggestions on European conferences about cell-based assays?I have a method for detecting protein interactions on living cells in real-time, which can be used for GPCR, targeted therapy, ADCs, cell-cell interactions etc. and want to find a suitable conference in Europe. It could be a method focused conference, but also within a biological area where my method can be applied. Any suggestions would be greatly appreciated.Thank you for all your suggestions! I will look into these.Following
- Stuart Gibb added an answer:Experience in transfecting with murine choroid plexus cells with Amaxa 4D Nucleofector?Hi, I am trying to stably transfect murine choroid plexus cells with the Amaxa 4D Nuclefector system. Does anyone have experience with this special celltype?Ive had great experience with the Amaxa 4D Nucleofector - I have transfected hippocampal neurons, MSCs and endothelial cells. Have you spoken to Lonza ?, they had great advice for me on specialized protocols
- Georgi Yordanov added an answer:Is an intact immune system essential for adequate cancer treatment?In another thread (http://tinyurl.com/k2em55e) we learn that there are two camps on approaches to cancer therapy: one is for targeted therapy, the other is phenotype-oriented. But both camps seems to agree that the immune system plays a pivotal role in both etiology and therapy. How is the immune system involved in etiology and can it assist in cancer therapy?An interesting article relating infectious disease, cancer and inflamation with stress-induced immune dysregulation could be found here:
It is known that chronic stress could compromise the immune system, which could increase the cancer incidence.
I think that the immune system is essential for adequate cancer treatment, but unfortunately most cytostatics are powerful immune-suppressors.
On the other hand, if so, one may ask the question if all experiments intended to test various cancer treatments on immuno-defficient mice are adequate?Following
- Julia Escandon 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?Gregory, Pritha: what phospho histone H3 antibody are you using? I am trying to also sort out my population of 4N cells but the cyclin B1 expression is too high in all of the cells, I think I am going to give a try to the p'histone... any suggestion of your preferred antibody?Following
- Jing Liu asked a question:Can anyone provide some references about GS-CHO cell stability?Recently, I should do some research about GS-CHO cell stability and need some reference about this topic. Thank you!Following
- Ayhan Bilir added an answer:How should I go about plating SH SY5Y cells in a 96 well plate?Hi all,
I am hoping someone has tips on how I should plate my SH SY5Y cells into a 96 well plate as I have limited experience with these smaller wells. Is 5000 cells an adequate amount to plate? And if I leave it for say 24hrs, would the cells not become overgrown by then?
Any current protocols that work would be much appreciated.University of Zirve Gaziantep City/Turkey
1.SHSY5Y cells has very much adhesion ability.
2. Numbers of cells seeding are very important
and 5000 cells are hıgh for each well of 96 well plate
3.The volume of medium for each cells for atachement ang growth is also important
4. Seeding cell density can change proliferation rates depending on tıme.Following
- Prasanta K Ray 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?In cancer, genetic material often unevenly divide producing different sets of cells, having also different properties; while some may not survive but many will. So from the standpoint of cancer cell killing one strategy to target one sets of cells may not also prove to be successful. Such mixed genetic behaviour has to be taken into consideration in cancer treatment strategy.Following
- Arnoud Boot added an answer:How to avoid non specific band in methylation specific PCR and to get the desired band?I am doing methylation specific PCR for various genes. I did bisulphite treatment referring protocol by Herman et al. In my methylation specific PCR result for all the genes ,I am not getting the desired band , but for all the genes , a band near 50 bp is coming. I don't know why is it happening?can any body tell me why is this occuring? and what can I do for its troubleshooting to get the desired product and to avoid this nonspecific band? I did simple PCR to check whether this is a primer dimer, but it was not primer dimer .Hi Lalita,
I agree with you that the enzyme degradation is unlikely to be the cause of the problem.
I think the concentration of your bisulfite converted DNA is fine, but this has no information value regarding efficiency of bisulfite conversion.
Good luck with your experiments, I've also tried using somebody elses primers a few times, but never got them to work...Following
- Nand kishor Roy added an answer:Can I use a normal genomic DNA isolation kit for isolation of apoptotic DNA of cancer cell?We have MN kit in our lab nucleo spin, but I want to know whether it can be used for isolation of apoptotic DNA.thank you Pradyumna Mishra Sir for your kind reply.Following
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