Cellular Biology Techniques

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How do I check tissue viability with MTT assay?

Imbert and Cullander already published a method in 1999 to investigate tissue viability by MTT assay. The authors said tissue samples were incubated in 2 mL of 2-mg/mL MTT (dissolved in PBS) for 2...

04 April 2015 8,864 1 View

Is HCl treatment bad for detection of nuclear protein?

As HCl pre-treatment is carried out for BrdU staining in tissue sections and it disrupts the chromatin structure, will it cause any problem if we want to co-stain with any nuclear antigen marker?

04 April 2015 1,634 2 View

Which viability assays can I do to confirm chondrogenic differentiation from MSC pellets?

Which viability assays can I do to confirm chondrogenic differentiation from MSC pellets? Cells were pelleted in micro centrifuge tubes and differentiated.

31 March 2015 8,025 6 View

Does anybody have experience with TGF-beta 1 - induced migration of HT-29 cells?

We have grown HT-29 cells (serum free) on non-coated transwells (8 um pore size, Greiner Bio) and incubated these at the basolateral side with 2.5, 5 and 10 ng/ml TGF-beta 1 for 12-24-48-72 hrs....

31 March 2015 925 11 View

Does anyone know if PPARg is expressed by wild type C2C12 myoblast?

I would like to induce trans-differention of C2C12 myoblast into adipocyte, and I've noticed that this porcess is PPARgamma-driven. Some colleagues, inducing this transdifferentiation, insert...

30 March 2015 9,212 2 View

What is the correct protocol for using TMRE in 96-well plate?

Dear colleagues, I am trying to determine mitochondrial membrane potential with the help of TMRE but it's not working. I am following the protocol from abcam website which is incubating the cells...

28 March 2015 4,055 4 View

What is a standard protocol for chicken embryonic myoblast culture (CEM)? How can I know that those are myoblasts or fibroblasts?

I was using 10th day embryo, DMEM 450 ml, 10% FBS 50 ml, streptomycin 5 ml, tripsin for CEM culture. Three times I failed due to contamination. Finally, I got good cell growth in all the six well...

26 March 2015 3,017 4 View

How can I avoid fungal growth in a primary cell culture under high laminar flow?

I have tried everything, high purity sterile media, adding fungizone, sterilizing the flow devices, the incubator, the incubator water, but I still can't shake off the fungal infection I get only...

26 March 2015 1,274 6 View

How is MitoTEMPO treatment given? Added along with the stress inducer or separately after the assay?

I am treating C2C1 myotubes with free fatty acid and would like to measure mitochondrial specific super-oxide production. I will be using MitoTEMPO for specificity. I have couple of questions...

11 March 2015 2,333 18 View

Can anyone tell me the difference between the isolation of enzymes from mitcochondria and peroxysomes?

05 March 2015 2,905 3 View

Has anyone successfully repeated the experiment of chemical induced PSC through small molecules published by Prof.Deng HK?

I have done this Cips experiment twice using classic 7 compounds in paper，only showing morphology change of MEF and cloning，no Oct4 activation，no Nanog activation，only showing the reactivation of...

03 March 2015 1,145 0 View

What is the best way to prove interaction between two proteins?

I am trying to carry out experiments which will prove the interaction between two different proteins. One protein is present in the cytoplasm and is thought to be secreted into the environment,...

03 March 2015 4,000 8 View

Will pre-treating petri dishes with collagen or gelatin affect HUVEC gene expression?

I'm trying to interface between two phases of a project - the first set of transcriptomic measurements were collected from cells plated on dishes that were not pre-treated. The next phase involves...

03 March 2015 6,476 8 View

Can we fix cells (1% PFA) before performing MACS cell separation?

I am doing single cell isolation from tumor mass (using collagenase D & DNAse I) for FACS analysis. I also have 1 step for MACS cell separation after collecting single cell suspension....

03 March 2015 5,833 7 View

How can we discuss about the results when both cell viability and mortality decreased?

Dear friends I have cultured hella cells and the cell mortality and viability have been checked under treatment with special drug. So, I have seen that the cell mortality and viability decreased...

03 March 2015 790 8 View

Can anybody suggest a method to estimate somatic cell count in colostrum?

I want to estimate SCC levels in colostrum (1st 2 days) of cow. I am facing difficulty due to thick consistency and presence of fat globules in colostrum. does anyone has experience of estimating...

03 March 2015 4,665 17 View

What are the points I should consider while designing the ligand receptor assay on an adherent cell line using flow cytometry?

The hormone binding reaction was initiated by addition of fluorescent tagged natural ligand to each in 6 well plate (4 to 5 lac cells/ per well 6 well plate). Incubations were performed in...

03 March 2015 7,143 4 View

Can I reuse polycarbonate tubes for exosome isolation?

I want to reuse the polycarbonate tubes for isolating exosomes. However i can see some sucrose stuck to the bottom of the ultracentrifuge tube from the previous isolation which is not getting...

03 March 2015 1,425 7 View

What is the optimum time to stimulate PBMCs?

what is the optimum time to stimulate PBMCs. i have gone through various manuscripts and they have mentioned various time frames ranging from 2-48 hours. i also came across many manuscripts which...

03 March 2015 5,485 7 View

How can I measure mitochondrial respiration in H9c2 cells?

I'm been trying to measure mitochondrial respiration of the cell line H9c2. I think i been having problem with the permeabilization. I wish to do de analisis using a Clark electrode. Thank...

03 March 2015 4,922 6 View

Any suggestions on apoptosis and cell cycle analysis, when I get no cells but fragments?

i'm studying apoptosis and cell cycle arrest induced by a drug, the problem is with some concentrations, after centrifugation at 300g/10min, in the cytometer i've plus than 1000 events/second wich...

03 March 2015 6,511 3 View

When do I apply a drug in colony formation assays?

When you do soft agar colony formation assay, do you treat (with drug/ modulator) cells growing on agar or you pre-treat the cells for a particular incubation period and then seed the treated...

03 March 2015 4,796 5 View

Should the Gibson Assembly be hot started?

The Gibson Assembly Mix is assembled on ice for obvious reasons, including not chewing back those ends too far. However, is there a suggestion to go into a machine pre-warmed to 50 degrees C or...

03 March 2015 8,256 1 View

What is the best method to lyse bovine kidney cell line?

I want to perform Co-IP from the bovine kidney cell line transfected with the Myc-tagged bait and HA-tagged prey proteins. I am using Pierce RIPA lysis buffer for the cell lysis but the lysate I'm...

03 March 2015 2,808 7 View

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